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Fig. 1. Transcriptional co-activator p75 interacts with JPO2. (A) Reciprocal co-immunoprecipitations of Flag-JPO2 and HA-p75. 293T cells co-transfected with HA-p75 and Flag-JPO2 or Flag-Mob2 expression constructs were lysed 24 hours post-transfection. Proteins immunoprecipitated (IP) with anti-Flag (left panel) or anti-HA (right panel) antibodies were detected by western blotting (WB). The migration positions of molecular mass standards are indicated to the left of each panel. (B) Flag-JPO2 complexes with endogenous p75. Lysates of 293T cells transfected with Flag-JPO2 were immunoprecipitated with anti-Flag or anti-p75/p52 antibodies. (C) Interaction between endogenous JPO2 and p75 proteins. Extracts of non-transfected 293T cells were immunoprecipitated with monoclonal anti-p75/p52 antibody, and JPO2 was detected using rabbit anti-JPO2 antibodies. The asterisk indicates a nonspecific protein detected during western blotting. (D) Overexpression of p75 post-transcriptionally augments the steady-state level of Flag-JPO2 protein. 293T cells were transfected with variable amounts of pBHA-p75 (lanes 2 and 6, 1 µg; lanes 3 and 7, 2 µg; lanes 4 and 8, 3 µg) with Flag-JPO2 (lanes 1-4) or Flag-Mob2 (lanes 5-8). Lanes 1 and 5, pBHA-p75 was omitted from transfection. The total amount of DNA in each transfection was adjusted to 4 µg using pcDNA6.V5HisB (Invitrogen). Cells were harvested 24 hours post-transfection, and 15 µg of total cellular protein was analyzed by SDS-PAGE and western blotting.
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