Figure 6. Expansion of the RABP17/E12 Interaction Network to Include Closely Related Proteins.

(A) The indicated combinations of plasmids including E12, MyoD and RANBP16 and the E-box pLuc reporter construct were transfected into HeLa cells and cell lysates analyzed for luciferase activity. Value for transfection of E-box pLuc only was set to 1 (column 1). *; p< 0.01 for the indicated bracketed pairwise comparisons and for the respective data value vs. that of column 1. (B) Regulation of endogenous p21^Waf1/Cip1 transcript level by RANBP16. Q-PCR analysis of RNA harvested from transfected 293T cells with *, p< 0.01 compared to empty vector. Representative of three independent transfection studies is shown. (C) The indicated combinations of plasmids including E47, MyoD, RANBP17 and the E-box pLuc reporter construct were transfected into HeLa cells and cell lysates analyzed for luciferase activity. For A and C, data is representative of three wholly independently conducted experiments, with transfections carried out in triplicate for each such independent experiment. Data are shown as the mean ± S.D. (D) Co-immunoprecipitation analysis reveals interaction of E12 with RANBP16 and E47 with RANBP17. Various combinations of plasmids as indicated by “+” signs at lane headings were transfected into COS cells. Cell lysates were subjected to immunoprecipitation (IP) and Western blot (WB) analysis with the indicated antibodies. 1/20 of total protein (2.5 μg) was analyzed for Western blot (INPUT). Equivalent protein content per sample was verified by Coomassie blue staining of 20 μg sample aliquots. For comparison purposes, the far right lane shows interaction of RANBP17 with E12, carried out in the same experiment and analyzed on the same Western blot as the other combinations presented in this panel.