Recently, Plzf has been found reference 2 to inhibit neurogenesis in Zebrafish. Taken together, Plzf has been implicated in hematopoietic, spermatogonial Inhibitors,Modulators,Libraries stem cells mainten ance and in inhibition of neurogenesis. Here we demonstrated a physical and functional inter action between Znf179 and the Plzf. Plzf altered the sub cellular localization of Znf179. Additionally, Znf179 regulated the protein levels of Plzf. Our findings provide possible function of Znf179 and highlight a potential re search direction for studying the molecular functions of Znf179. Methods Plasmid construction A PCR fragment encoding the N terminal of Znf179 was subcloned into vector pBTM116 in frame with LexA to generated the LexA Znf179 bait. pGal AD Plzf deletion mutants were engineered by subcloning PCR amplified Plzf fragments into the yeast vector pACT2, which expresses the Gal4 activation domain.
To gene rate Znf179 and Plzf expression vectors for Inhibitors,Modulators,Libraries mammalian cells, the full length or partial cDNA fragments were ampli fied by PCR using IMAGE clone 4506141 and 4944546 as templates, respectively. Sequences of the primers used were listed in Additional file 1, Table S1. EGFP Znf179, EGFP Znf179 and EGFP Znf179 were generated by inserting Znf179 cDNA fragments into pEGFP vector. Flag Plzf, Flag Plzf, Flag Plzf, Flag Plzf, Flag Plzf and Flag Plzf were generated by inserting Plzf cDNA fragments into pCMV Tag2 vector. The full length cDNA fragments of Znf179 and Plzf were also inserted in frame into the pM vector, a vector for the expression of GAL4 DBD fusion proteins from a constitutive SV40 early promoter.
GSK-3 The constructs of HA Plzf and Arora kinase C promoter were described elsewhere. pFR Luc reporter plasmid contains a synthetic pro moter with five tandem repeats of the yeast GAL4 binding elements that Inhibitors,Modulators,Libraries control expression of the firefly luciferase gene. pRL TK, a plasmid contains the Renilla luciferase as transfection control, was purchased from Promega. Yeast two hybrid screen and B galactosidase activity assay The LexA Znf179 construct was used to screen against with mouse brain cDNA library. Yeast two hybrid screen was performed as described previ ously. L40 yeast strain was first transformed with LexA Znf179, followed by 100 ug of the brain cDNA library transformation. The library of transfor mants was selected on medium lacking histidine, leu cine, and tryptophan.
His colonies were further tested for B galactosidase activity using Inhibitors,Modulators,Libraries a colony lift filter assay. The plasmids from both of His and X gal col onies were isolated by the curing process of MC1066 bacterial strain and retransformed with LexA read more Znf179 or LexA lamin to test the binding specificity. The library plasmids conferred that the Znf179 specific interactions were then subjected to DNA sequence ana lysis. Quantitative X gal assays were performed with yeasts containing pairs of bait and prey plasmids as indicated.