P311 binds to the PPARγ2 promoter and it is an intrinsically disordered protein.

A. Western blot analysis of chromatin immuno-precipitates (ChIP) of myc-P311 in pcP311 and empty vector (pCMV) transfected 3T3-L1 adipocytes (10 days post induction). The bands represents myc-P311 expression and pulldown (upper panel). qPCR was used to measure PPARγ2 promoter amplification to examine P311 recruitment to PPARγ2 promoter in ChIP complexes in P311 over expressed adipocytes (pc+ and pcP311+) and empty vector transfected controls (lower panel). B. Western blot analysis of ChIP of RNApolII in pcP311 and pCMV transfected 3T3-L1 adipocytes. Uninduced preadipocyte lysates and ChIP samples (pc- and pcP311-) had basal levels of RNApolII whereas adipocytes (pc+ and pcP311+) showed elevated levels of RNA RNApolII (upper panel). qPCR was used to measure PPARγ2 promoter amplification to examine RNApolII recruitment to PPARγ2 promoter in ChIP complexes in P311 over expressed adipocytes empty vector transfected controls (lower panel). C. Secreted-pair dual luminescence assay showed the binding of c-myc-P311 to the PPARγ promoter compared to the empty vector promoter (NEG) and empty vector control (pCMV). D. Predicted structure of monomeric P311. N- and C-represents N- and C-termini of proteins. E. Predicted structure of trimeric P311. F. Western blot analysis showed the overexpression of monomeric (8 kDa) and trimeric his-tag-P311 (~24 kDa) in E. coli (BL21D3) cell lysates immunodetected with P311-antibody. G. Western blot analysis showed the overexpression of monomeric and trimeric c-myc-tag-P311 immunodetected with c-myc-tag-antibody in 3T3-L1 preadipocytes and adipocytes. pET-P311 and pcP311 are bacterial and mammalian expression plasmids of his-tagged and myc-tagged P311 respectively. Empty vectors, pET and pcP311 served as controls.