Fig. 4.

Hexim1 sequesters P-TEFb from CIITA on the endogenous HLA-DRA promoter. (a) Levels of f:CIITA.IF1 and x:Hex1 proteins in HeLa cells. f:CIITA.IF1 (1 μg) was expressed alone or with x:Hex1 (1 μg) in HeLa cells. The expression of these proteins was determined by Western blotting with αCIITA and αXpress antibodies (Top and Middle). Levels of GAPDH were used to assess inputs of proteins for immunoblotting (Bottom). (b) ChIP assays were carried out with αCIITA, αCycT1, and αRNAPII antibodies. Sequences corresponding to the endogenous HLA-DRA promoter were detected by PCR. Additionally, the promoter for GAPDH gene was used as the negative control. Presented above the analyses is a diagrammatic representation of the HLA-DRA promoter and gene. Arrows below the diagram define positions of primers used in ChIP assays. Antibodies used in ChIP analyses are named next to the gels. (c) All results for PCR analyses were in the linear range of PCR. Amplifications with different numbers of cycles were carried out with αCycT1 and αCIITA immunoprecipitates. Numbers to the left depict actual number of cycles used in PCR.