Figure 6.

GR competes with RelA for PKAc association in vivo. (A) GR blocks RelA:PKAc protein association in vivo. Human 293T cells were transfected with 5 μg of CMVRelA, 5 μg of pcPKAc, and 5 μg of CMXGR or empty vector, as indicated. Cells were mock-treated or activated with 1 μM Dex for 12 h and harvested 48 h after transfection. (AI) Cell lysates were immunoprecipitated with 5 μl (1 μg) of RelA sc-109 rabbit polyclonal antibody (lanes 3–6) or with the same amount of normal rabbit IgG as control (lane 2). Immunoprecipitates were analyzed by Western blot with PKAc-specific antibody. WCE (lane 1) were loaded in parallel with the immunoprecipitates to show comigration. (AII) Quantitation of PKAc coprecipitated by RelA-specific antibody. The y axis shows the ratio of PKAc to IgG. Quantitation is performed with NIH image program. (AIII) Similar amount of WCE from cells corresponding to lanes 3–6 in AI was immunostained with GR-specific antibody (sc-1002). (AIV) The same blot shown in AIII was stripped and reprobed with RelA-specific antibody. (B) GR represses PKAc-dependent NF-κB transactivation. CV1 cells were cotransfected with 120 ng of Igk3-Luc reporter construct, 75 ng of CMXβgal, 33 ng of CMV RelA, and increasing amount of pcPKAc expression vector, as indicated. The RSVGR and RSVGRΔ589–697 were used at 3:1 M ratio with RelA. All of the transfection points were equalized for the total amount of expression vectors. The y axis shows fold activation as measured by Igk3-driven Luc activity. The histogram is representative of a triplicate experiment.