Fig. 4: REGγ interacts with PP2Acα and directs its degradation.
From: The proteasome activator REGγ accelerates cardiac hypertrophy by declining PP2Acα–SOD2 pathway
a REGγ knockout increases of PP2Acα protein levels in mice heart, and b REGγ knockdown upregulates PP2Acα protein levels, and c REGγ overexpression downregulates PP2Acα protein levels in AC16 cells by immunoblotting analysis, whereas d REGγ knockout or knockdown downregulates PP2Acα mRNA expression in murine heart or AC16 cells. e Interaction between REGγ and PP2Acα in 293T cells was determined by coimmunoprecipitation and immunoblotting analysis following transient transfection of Flag-REGγ/Flag vector and HA-PP2Acα into 293T cells. f Reciprocal interaction between REGγ and PP2Acα was performed by coimmunoprecipitation as indicated following transient transfection of Flag-PP2Acα/Flag vector and HA-REGγ into 293T cells. g Endogenous REGγ in AC16 cells was precipitated using anti-REGγ antibody or with IgG control, and coprecipitated PP2Acα was detected by immunoblotting. h Reciprocal interaction between endogenous REGγ and PP2Acα in AC16 cells was performed by using anti-PP2Acα antibody or IgG control, and coprecipitated REGγ was detected by immunoblotting. Stability of endogenous PP2Acα in (i) siNeg and siREGγ NRCMs and (j) corresponding quantitation graphs of relative PP2Acα degradation, or (k) siNeg and siREGγ AC16 cells and (l) corresponding quantitation graphs of relative PP2Acα degradation. (The experiments were repeated three times; error bars represent standard deviation, *P < 0.05, **P < 0.01, Student’s t test.) Cells were treated with CHX (100 μg/mL) for indicated times followed by immunoblotting. Stability of endogenous PP2Acα in (m) siNeg, siREGγ, and siREGγ plus GFP-REGγ plasmid AC16 cells and (n) AC16 cells pretreated with MG132. Cells were treated with CHX (100 μg/mL) for indicated times followed by immunoblotting. o REGγ directly promoted the degradation of PP2Acα. In vitro proteolytic analyses were performed using purified REGγ, 20S proteasome, and in vitro translated PP2Acα protein as indicated and described in “Materials and methods.” A known substrate of REGγ, p21, was shown as a positive control.
