We collected samples from 138 individuals Buparlisib (97 adults and 41 children) on cART with virological, immunological or clinical signs of treatment failure.

HIV-1 pol sequences were obtained using an in-house method. Resistance mutations were identified according to the 2007 International AIDS Society (IAS)-USA list and predicted susceptibility to cART was scored using the anrs algorithm. Resistance mutations were detected in 112 patients (81%), 74% in adults and 98% in children. Triple-, dual- and single-class drug resistance was documented in 27%, 43% and 11% of the study subjects, respectively. Multiple logistic regression showed that resistance was independently associated with type of treatment failure [virological failure (odds ratio (OR)=1) vs. immunological failure (OR=0.11; 95% confidence interval (CI) 0.030–0.43) vs. clinical failure (OR=0.037; 95% CI 0.0063–0.22)], route of transmission (OR=42.8; 95% CI 3.73–491), and years on therapy (OR=1.81; 95% CI 1.11–2.93). The prevalence of antiretroviral resistance was high in Honduran HIV-infected patients with signs of treatment failure. A majority of study subjects showed dual- or triple-class resistance to nucleoside reverse transcriptase

inhibitors, nonnucleoside reverse transcriptase inhibitors and protease inhibitors. Virologically defined treatment failure was a strong predictor of resistance, indicating BAY 80-6946 mw that viral load testing is needed to correctly identify patients with treatment failure attributable to resistance. The mortality of HIV-1 infection has decreased dramatically in the developed parts of the world following the introduction of combination antiretroviral therapy (cART) in 1996 [1–3]. cART typically involves therapy with two nucleoside reverse transcriptase inhibitors (NRTIs) and a protease inhibitor (PI) or a nonnucleoside L-NAME HCl reverse transcriptase inhibitor (NNRTI) [3,4]. Considerable efforts are being made to improve access to cART in developing countries. It is estimated that more than 9 million adults

in low- and middle-income countries with advanced stages of HIV infection are in urgent need of cART. By December 2007, only about 3 million of these patients were actually receiving therapy. Currently, it is estimated that 390 000 individuals (62%) of those in medical need of cART in Latin America and the Caribbean are provided with medication by established treatment programmes [5]. Honduras is estimated to have one of the highest HIV-1 prevalences (0.7%; range 0.4–1.4%) in Latin America [6]. Of the large number of HIV-positive individuals, 12 000 are estimated to be in need of cART (Table 1). The National HIV/AIDS Program in Honduras began to scale up access to therapy in 2002, and since then many patients have gained access to cART. At present approximately 6000 patients have been under treatment, of whom around 700 have interrupted therapy and more than 800 have died [7].

In women who have a detectable VL it may be possible to optimize their HAART regimen to reduce the risk of MTCT (See Recommendation 4.2.6). 7.3.5 The management of PPROMs at ≥34 weeks is the same as term ROM (see Section 7.3 Management of spontaneous rupture of membranes) except women who are 34–37 weeks’ gestation learn more will require group B streptococcus prophylaxis in line with national guidelines. Grading: 1C 7.3.6 When PPROM occurs at <34 weeks:

Grading: 1C Intramuscular steroids should be administered in accordance with national guidelines. Virological control should be optimized. There should be multidisciplinary discussion about the timing of delivery. There are no data to inform the optimum management of preterm labour or early preterm pre-labour ROMs. Decisions regarding the optimum management of early preterm ROM require the assessment of a number of

factors, including the exact gestation, facilities available, maternal VL and presence of other co-morbidities such as infection and pre-eclampsia. Corticosteroids to improve fetal lung maturation should be given as per the Royal College of Obstetricians Selisistat mw and Gynaecologists guidelines [49] and (if delivery is to be delayed) oral erythromycin [50]. Decisions regarding timing of delivery should be made in consultation with the full MDT, including the neonatal unit. There is no evidence that steroids for fetal lung maturation (with the associated 24-h delay in induction) are of overall benefit at 34–37 weeks’ gestation in women with ROMs, thus delay for the optimization of fetal lung maturity is not recommended. buy Baf-A1 For this reason, and to minimize the risk of developing chorioamnionitis, induction is recommended from 34 weeks’ gestation in women with ROMs who are not in labour. If the maternal VL is not fully suppressed, consideration should be given to the options available to optimize therapy. An additional concern is that the early preterm infant

may be unable to tolerate oral therapy and therefore loading the infant through the transplacental route with maternal therapy is recommended (see Section 5: Use of antiretroviral therapy in pregnancy). There is most experience with maternal oral nevirapine 200 mg stat >2 h before delivery, but double-dose tenofovir and standard-dose raltegravir can also be considered. 7.4.1 Intrapartum intravenous zidovudine infusion is recommended in the following circumstances: For women with a VL > 10 000 HIV RNA copies/mL plasma who present in labour, or with ROMs or who are admitted for planned CS. Grading: 1C For untreated women presenting in labour or with ROMs in whom the current VL is not known. Grading: 1C In women on zidovudine monotherapy undergoing a PLCS intravenous zidovudine can be considered. Continued oral dosing is a reasonable alternative.

13 ± 5.84; CS−, 15.35 ± 5.97; t32 = 2.12, P = 0.042). No significant differences between conditions were present before affective learning (CS+, 16.62 ± 6.82; CS−, 17.45 ± 6.67;

t32 = −1.06, P = 0.300). In addition, we tested for effects of relatively increased CS− as compared to CS+ processing within a mirror-symmetric frontal region in the left hemisphere, as well as for differential effects across hemispheres. While there was no significant Session × Valence interaction in the left hemisphere (P > 0.05), the three-way interaction with Hemisphere marginally reached significance (F1,32 = 3.62, P = 0.066). The localisation of the above analysed effects fitted www.selleckchem.com/products/lgk-974.html our expectations, as regions for sensory auditory processing and areas Ibrutinib mw in parietal and frontal cortex as part of a distributed attentional network were highlighted in the analysis. Though unexpected, one further

neural generator cluster at the right occipitocerebral junction (the spatial resolution of the MEG in combination with the applied head and conductivity models does not allow a more distinct localisation of effects) showed a significant Session × Valence interaction (F1,32 = 8.02, P = 0.008) with relatively increased CS+ compared to CS− processing. Interestingly, this area also reveals an interaction with Hemisphere (F1,32 = 9.3, P = 0.005) when compared to a corresponding left hemispheric region although the relatively increased CS− processing in the left hemisphere was not significant. To summarise the MEG data, we found an affect-specific modulation of the event-related fields that were recorded in response to multiple click-like tones before and after MultiCS conditioning: in the pre- vs. post-conditioning comparison, the emotion effect was strongest between 100

and 150 ms after CS onset within a left-hemispheric posterior sensor cluster with relatively stronger RMS amplitudes for CS− as compared to CS+ processing. Source localisation for this time-interval overlapping the auditory N1m revealed that the presence Glutathione peroxidase of emotionally salient stimuli affected auditory processing mainly in two neural generator clusters in the left parietotemporal and the right prefrontal cortex. The data suggested that aversively conditioned tones were preferentially processed in the right hemisphere, while unpaired CS evoked stronger brain activation in the left hemisphere. For the parietotemporal region, this assumption was statistically supported by an interaction of the emotion effect with hemisphere. For the frontal source cluster, a trend pointed towards the same interpretation. Contrary to our assumptions, the presence of shock-conditioned tones did not significantly modulate AEFs in the earlier P20–50 m time-interval.

All data are expressed as the means and standard deviations of three determinations per experimental condition. Statistical significance was determined using a one-way anova followed by Dunnett’s multiple comparison test. A P-value < 0.05 was considered statistically significant. The T3SS-associated chaperone and the effector complex

bind to each other with high affinity (Luo et al., 2001). Therefore, we used selleck products a screening assay using T3SS2 effectors fused with GST to pull down chaperone candidates. The amino-terminal regions of T3SS2 effectors (VopC, VopL, VopP, and VopT) fused to the CyaA (Bordetella pertussis toxin) catalytic domain can be injected into host cells (Kodama et al., 2007) (T. Kodama, unpublished data). This is consistent with other T3SS effectors and suggests that the amino-terminal regions of V. parahaemolyticus T3SS2 effectors are sufficient for efficient secretion and translocation. In general, amino-terminal domains (1–200 amino acids) of T3SS effectors contain the amino-terminal secretion signal of the T3SS and the chaperone-binding domains, which are both essential for effector secretion

(Feldman & Cornelis, 2003; Parsot et al., 2003). Plasmids expressing the PARP inhibitor trial amino-terminal domains (1–200 amino acids) of the T3SS2 effectors VopC, VopL, VopP, and VopT fused to GST were introduced into V. parahaemolyticus knockout strains for each gene. The GST fusions expressed in V. parahaemolyticus strains were purified using glutathione beads and separated using SDS-PAGE. The molecular weights of most T3SS-associated chaperones are less than 20 kDa (Feldman & Cornelis, 2003;

Parsot et al., 2003); therefore, the areas containing proteins of these molecular weights were carefully observed. Although the T3SS2 effectors fused to GST appeared to be unstable (a lower amount of T3SS2 effector fusions than breakdown products was observed), the amino-terminal 1–200 amino acids of the T3SS2 effectors fused to GST were copurified with a specific band that was not observed in the negative control (GST alone), as shown in Fig. 1a. Mass analysis revealed proteins interacting with GST–VopC1–200, GST–VopL1–200, and GST–VopT1–200 (Fig. 1b), while GST–VopP1–200 did not interact with any specific proteins that could be chaperone candidates. The results suggested that only one protein encoded much in the Vp-PAI, VPA1334 (designated VocC; Vop chaperone C), appeared to be a T3SS chaperone candidate. The molecular weight and the isoelectric point of VocC were estimated as 14.3 kDa and 5.41, respectively. Based on the information from previously identified T3SS-associated chaperones (Feldman & Cornelis, 2003; Parsot et al., 2003), these values indicate that VocC is a possible T3SS2-associated chaperone for VopC, VopL, and VopT, and this result may categorize VocC as a type IB class chaperone, which chaperones multiple effectors (Parsot et al., 2003).

All data are expressed as the means and standard deviations of three determinations per experimental condition. Statistical significance was determined using a one-way anova followed by Dunnett’s multiple comparison test. A P-value < 0.05 was considered statistically significant. The T3SS-associated chaperone and the effector complex

bind to each other with high affinity (Luo et al., 2001). Therefore, we used HDAC inhibitor a screening assay using T3SS2 effectors fused with GST to pull down chaperone candidates. The amino-terminal regions of T3SS2 effectors (VopC, VopL, VopP, and VopT) fused to the CyaA (Bordetella pertussis toxin) catalytic domain can be injected into host cells (Kodama et al., 2007) (T. Kodama, unpublished data). This is consistent with other T3SS effectors and suggests that the amino-terminal regions of V. parahaemolyticus T3SS2 effectors are sufficient for efficient secretion and translocation. In general, amino-terminal domains (1–200 amino acids) of T3SS effectors contain the amino-terminal secretion signal of the T3SS and the chaperone-binding domains, which are both essential for effector secretion

(Feldman & Cornelis, 2003; Parsot et al., 2003). Plasmids expressing the buy GDC-0980 amino-terminal domains (1–200 amino acids) of the T3SS2 effectors VopC, VopL, VopP, and VopT fused to GST were introduced into V. parahaemolyticus knockout strains for each gene. The GST fusions expressed in V. parahaemolyticus strains were purified using glutathione beads and separated using SDS-PAGE. The molecular weights of most T3SS-associated chaperones are less than 20 kDa (Feldman & Cornelis, 2003;

Parsot et al., 2003); therefore, the areas containing proteins of these molecular weights were carefully observed. Although the T3SS2 effectors fused to GST appeared to be unstable (a lower amount of T3SS2 effector fusions than breakdown products was observed), the amino-terminal 1–200 amino acids of the T3SS2 effectors fused to GST were copurified with a specific band that was not observed in the negative control (GST alone), as shown in Fig. 1a. Mass analysis revealed proteins interacting with GST–VopC1–200, GST–VopL1–200, and GST–VopT1–200 (Fig. 1b), while GST–VopP1–200 did not interact with any specific proteins that could be chaperone candidates. The results suggested that only one protein encoded Y-27632 2HCl in the Vp-PAI, VPA1334 (designated VocC; Vop chaperone C), appeared to be a T3SS chaperone candidate. The molecular weight and the isoelectric point of VocC were estimated as 14.3 kDa and 5.41, respectively. Based on the information from previously identified T3SS-associated chaperones (Feldman & Cornelis, 2003; Parsot et al., 2003), these values indicate that VocC is a possible T3SS2-associated chaperone for VopC, VopL, and VopT, and this result may categorize VocC as a type IB class chaperone, which chaperones multiple effectors (Parsot et al., 2003).

Three colonies from an M1 pure culture plate were initially vortexed with 50 μL lysing solution (0.05% sodium dodecyl sulfate; 30 mM NaOH). Following incubation for 15 min at 95 °C and brief centrifugation, the solution was diluted with 450 μL H2O and centrifuged for 15 min. this website In addition to 2.0 μL alkaline lysis supernatant as the template, the 50-μL PCR mixture contained 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 0.1% Triton X-100, 1.2 mM MgCl2, each dNTP (0.1 mM each), 0.2 μM of each primer, and 0.625 U of Perpetual OptiTaq DNA polymerase (Roboklon, Berlin, Germany). Amplification was performed using the following conditions: 95 °C for 2 min, followed by 33 cycles of 95 °C for 20 s, 55 °C for 30 s, and 72 °C for 1.5 min, with a final step

of 10 min at 72 °C. The PCR product was sequenced by SMB (Berlin, Germany). Chromatogram sequences were trimmed using Chromas Lite (Technelysium Pty Ltd, Tewantin, Qld, Australia), alignments were performed using bioedit (Hall, 1999), and the phylogenetic tree was constructed using the neighbor-joining method in the arb program (Ludwig et al., 2004). Fe(II) oxidation experiments were performed using the bicarbonate-buffered, gradient-culture medium described above. To rule out the possibility that the growth of strain M1 was occurring on organic compounds in the agarose ABT-737 chemical structure gel,

we designed an experiment with three treatments (three replicates each). The first treatment utilized gradient vials with 50 mM FeCl2 in the lower layer. The second treatment excluded FeCl2 from the lower layer and the third treatment substituted 5 mM Na2S for FeCl2. The sulfide was used to establish a redox and O2 gradient in the vials in case the growth of M1 required microoxic conditions. In all cases, resazurin (0.0001%) was included in

the 15-mL, upper layer to allow visualization of the depth of O2 penetration. Inoculum was prepared by resuspending colonies from plates in 2 mL sterile many upper layer. Two 50-μL aliquots of the resultant suspension were used to inoculate gradient systems at a depth of about 1 cm below the upper-layer surface. Two additional vials containing FeCl2 in the lower layer were inoculated with only sterile upper layer and used as abiotic controls. All gradient vials were purged with N2 : CO2 for 10 s before closing the screw-caps to partially remove O2. Vials were incubated statically in the dark at room temperature. At the conclusion of the 8-day experiment, cells were counted by epifluorescence microscopic examination after staining cells with 4′,6-diamidino-2-phenylindole (DAPI) after fixation with 3.4% formaldehyde (Kepner & Pratt, 1994). Where necessary, iron oxide precipitates were removed before staining using an oxalate dissolution method as described elsewhere (Roden & Zachara, 1996). To determine the vertical distribution of cells in an iron-oxidizing, gradient-culture system, aliquots of an upper-layer suspension for DAPI counting were withdrawn at 5-mm depth intervals using a sterile syringe.

18%, respectively; OR 2.5; P<0.01) (Table 1). Those with CAC were more likely to have fatty liver disease than those without CAC (23%vs. 8%, respectively; OR 3.4; P<0.01). Regarding body measurements, the thigh circumference,

the physician visual assessments of body fat at six locations, and the percent of body fat as calculated by caliper measurements were univariately associated with CAC (Table 1). No other circumference or individual skinfold measurement was associated with CAC (data not shown). HIV-specific factors that were significantly associated with CAC in the univariate analyses included a longer duration of HIV infection (median 18 vs. 9 years for those with and without CAC, respectively; OR 1.1 per year; P<0.01), a lower CD4 nadir (184 vs. 285 cells/μL, respectively; OR 0.7; P<0.01) and current HAART use (93%vs. 78%, respectively; OR 4.0; P<0.01). The duration of exposure to each of the three main drug classes RAD001 was also positively associated with CAC in the univariate models. In addition, individual use (current or ever) of abacavir or ritonavir were each associated with CAC (Table 1). Current receipt of tenofovir, efavirenz or atazanavir

was not associated with CAC (data not shown). In the multivariate analyses, older age (OR 4.3 per 10-year increase; P<0.01), fatty liver disease (OR 3.8; P<0.01) and hypertension (OR 2.6, P<0.01) were significantly associated with the presence of coronary atherosclerosis as determined using the CAC score (Table 3). There were no significant associations with body measurements or HIV-specific factors, including antiretroviral medication Androgen Receptor Antagonist chemical structure use (evaluated as months of use, current use and ever use), in the multivariate model. Edoxaban The multivariate model was replicated excluding those with HCV seropositivity (n=6) with no significant differences noted in the association of fatty liver disease and CAC [OR 4.2; 95% confidence interval (CI) 1.6–11.1; P<0.01]. Finally, in order to evaluate the relationship of fatty liver disease and CAC independently of the metabolic syndrome, we repeated the model examining only participants without the metabolic syndrome (n=173);

fatty liver disease remained associated with a positive CAC score in this subset (OR 5.4; 95% CI 1.5–19.2; P<0.01). We performed sensitivity analyses to evaluate the robustness of our findings. As fatty liver disease can be caused by either NAFLD or alcohol overuse, we excluded patients with excessive alcohol use (n=12) and noted similar findings. As the risk factors for coronary atherosclerosis may vary by gender, we also performed the analyses among only male patients and found the same associations. Finally, using multivariate linear regression modelling, we evaluated associations with the CAC score as a continuous variable and found that age (coefficient 4.4; 95% CI 2.3–6.4, P<0.01) and fatty liver disease (coefficient 88.1; 95% CI 30.2–146.1; P<0.

Statistical evaluations were performed using spss 13.0 software. Repeated-measures anovas

(3 × 2) were run for syllables, words and sentences, with separate analyses for response accuracy and vocal reaction times (RTs). As RTs for syllables were already short at T0, for this group of stimuli RTs were not collected. For each analysis, two within-subject factors were included: Time (T0 vs. T10 vs. F/U) and Condition (real stimulation vs. sham). Interaction was explored using the Scheffé post hoc test. For each stimulus, vocal RT was measured from the onset of the participant’s response to the end of the stimulus production using Free Audio Editor 6.9.1 software. The analysis showed a significant effect of Time [Baseline (T0) vs. End of treatment (T10) vs. Follow-up (F/U), selleck F2,14 = 31.76, P = 0.000] MAPK inhibitor and Condition (Real Stimulation vs. Sham, F1,7 = 16.76, P = 0.005). The interaction of Time × Condition was also significant (F2,14 = 4.50, P = 0.031). The Scheffé post hoc test revealed

that, while no significant differences emerged in the mean percentage of correct syllables between the two conditions at T0 (differences between Real Stimulation and Sham, 2%; P = 1), the mean percentage accuracy was significantly greater in the real stimulation than in the sham condition, both at T10 (differences between Real Stimulation vs. Sham at T10, 27%; P = 0.027) and at F/U (differences between Real Stimulation vs. Sham at F/U, 24%; Branched chain aminotransferase P = 0.041). No significant differences emerged in the mean percentage accuracy between T0 and T10 for the sham condition (difference between T0 and T10, 12%; P = 0.603; see Fig. 3). The analysis showed a significant effect of Time (T0 vs. T10 vs. F/U; F2,14 = 38.93, P = 0.000) and Condition (Real

Stimulation vs. Sham; F1,7 = 7.88, P = 0.026). The interaction of Time × Condition was also significant (F2,14 = 4.46, P = 0.032). The Scheffé post hoc test revealed that, while no significant differences emerged in the mean percentage of correct words between the two conditions at T0 (differences between Real Stimulation and Sham, 7%; P = 0.541), the mean percentage accuracy was significantly greater in the real stimulation than in the sham condition both at T10 (differences between Real Stimulation and Sham at T10, 22%; P = 0.000) and at F/U (differences between Real Stimulation and Sham at F/U, 13%; P = 0.004; see Fig. 3). The analysis showed a significant effect of Time (T0 vs. T10 vs. F/U; F2,14 = 15.11, P = 0.000) and Condition (Real Stimulation vs. Sham; F1,7 = 6.76, P = 0.035). The interaction Time × Condition was also significant (F2,14 = 6.33, P = 0.011).

The common thread included in these definitions is use of immigrant status, race and/or ethnicity to classify individuals because the frequent view is that these factors predict a “complex set of behaviours.” Race and ethnicity, however, are poor predictors for

behaviors and/or health beliefs of individuals. In this increasingly mobile and culturally, ethnically, and racially intertwined world, a large number, perhaps a majority, of travelers cannot be classified on the basis of their immigrant status and ethnicity. It is rather essential that each individual’s preexisting Selleck Enzalutamide health knowledge and beliefs be assessed during a travel visit. Dr Arguin states that it is not a change in travel patterns, but rather a significant increase in the total number of travelers SB431542 that is occurring. We believe that there is a distinct evolution in the type of traveler being seen in travel clinics, and that this has prompted the discussion on the relevance of the traditional immigrant/racial/ethnicity-based

definition of the VFR traveler. The complexity in defining this group of travelers is probably the proverbial “tip of the iceberg,” because this is the first non-privileged travel population to seek pre-travel care routinely. It is likely that the disparities in morbidity and mortality patterns demonstrated in the literature, and experienced by this population, are more closely related to their socioeconomic status than to their immigrant status, race, and/or ethnicity. This issue has not arisen before as socioeconomic factors restricted this group from attending travel clinics. The paper by Leder and co-workers describing a decreasing gradient of adverse health outcomes from an “immigrant VFR” to “traveler

Rutecarpine VFR” to “tourist” is used by Dr Arguin as an argument that returning to one’s country of origin is a risk, independent of genetic factors or cultural background.4 This same paper, however, demonstrates that “nonimmigrant VFR travelers” (who are not identified using immigrant status, race, or ethnicity) exhibit an increased risk of adverse health outcomes. It is important to note that this latter group, reported by Leder, was by no means exclusively constituted by spouses and offspring accompanying an ethnic traveler. The complexity in defining travelers is increasing, as demonstrated by the case of a woman born and living in the United States who will be traveling to India with her Indian-born boy-friend to visit his family. Further, with Dr Arguin’s criteria (according to the current CDC definition) a person must be traveling from a higher-income to lower-income country to be a VFR traveler.

coli. However, hydrophobicity Panobinostat price profile analysis revealed that the N-terminus of the A domain has a putative transmembrane segment. The N-terminus of the A domain might act as an integral membrane anchor, indispensable for FtsY membrane association (Bibi et al., 2001). When this putative transmembrane segment was fused to the E. coli NG domain, the chimera construct was capable

of rescuing wild-type EcFtsY depletion in a conditional FtsY-deletion mutant of E. coli. In contrast, the E. coli NG domain alone could not fully rescue wild-type FtsY depletion (Maeda et al., 2008). These results suggest that the N-terminus of S. coelicolor FtsY (ScFtsY) has a functional role. The ScFtsY N-terminus may contribute to the membrane targeting of FtsY, but there is no direct evidence. In this study, the membrane-targeting ability of the N-terminal hydrophobic segment of the ScFtsY A domain was assessed by membrane protein extraction and Mal-PEG

labeling experiments. Results show that this part of the ScFtsY A domain might form a membrane insertion structure that can anchor ScFtsY to the membrane. The S. coelicolor strains used in this study are listed in Table 1. The E. coli strain ET12567 (MacNeil et al., 1992), which contains the plasmid pUZ8002, was used for plasmid introduction by conjugation into S. coelicolor M145 (Kieser et al., 2000). http://www.selleckchem.com/products/Romidepsin-FK228.html All S. coelicolor strains were grown at 30 °C, 220 r.p.m. min−1 in TSB liquid media for protein expression. Apramycin (50 μg mL−1) was added when necessary. All the plasmids used in this study are listed in Table 2. All primers are listed in Supporting Information, Appendix S1, and the detailed protocol for plasmid construction and protein expression is provided in Appendix S2. Subcellular fractions were isolated as described in the study by de Leeuw et al. (1997). Cells were suspended in lysis buffer (Mao et al., 2009) and lysed by freezing and short ultrasonic treatment. The cellular debris was removed from

the lysate by sedimentation (12 000 g for 15 min); the supernatant was then subjected to ultracentrifugation (356 000 g for 45 min), and the membrane pellet fraction [precipitant (‘P’)] was separated from the soluble fraction GPX6 [supernatant (‘S’)]. The supernatant was precipitated with 1 vol 10% TCA and resuspended in SDS-loading buffer, whereas the pellet fraction was directly dissolved in the same amount of SDS-loading buffer. The same amount of ‘P’ and ‘S’ samples was loaded onto an SDS-PAGE gel. The EGFP mutants in the samples were detected using the EGFP antibody. The protein content in ‘P’ and ‘S’ was calculated using the Quantity One software (Bio-Rad™). For carbonate extraction, the membrane pellet fraction, ‘P’, was incubated with 0.2 M Na2CO3 for 30 min at 4 °C and subsequently ultracentrifuged for 45 min at 356 000 g; the precipitant was the membrane pellet fraction (‘P′’), and the supernatant was the soluble fraction (‘S′’).