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Figure 3: α-actinin-4 K256E “chokes” the proteasome and exacerbates ER stress. (a) GFP-α-actinin-4 K256E undergoes ubiquitination. COS-1 cells were transiently transfected with GFP-α-actinin-4 wild type (WT) or K256E mutant (Mut), and the cells were incubated with the proteasome inhibitor, MG132. Lysates were immunoprecipitated with anti-GFP antibody (+), or nonimmune IgG (−) in controls. Then, the immunoprecipitates were immunoblotted with antibodies to ubiquitin (upper panel) or GFP (lower panel). (b, c) COS cells were transiently cotransfected with the GFPU proteasome reporter (or GFP for comparison), plus GFP-α-actinin-4 K256E or wild type. Lysates were immunoblotted with anti-GFP antibody. In COS cells transfected with α-actinin-4 wild type, expression of the GFPU reporter is substantially lower than GFP, since GFPU is readily degraded by the ubiquitin-proteasome pathway, but GFP is stable. Mutant α-actinin-4 did not affect GFP expression. GFPU expression was enhanced by cotransfection of α-actinin-4 K256E, indicating that this mutant impaired degradation of GFPU by the ubiquitin-proteasome system. (c) GFPU mutant versus wild type. (d, e) COS cells were transiently transfected with GFP-α-actinin-4 wild type or mutant. Lysates were immunoblotted with antibodies to GFP, grp94 or CHOP. NS, nonspecific band-loading control. , mutant versus wild type. The figure is adapted from [35] with permission of the American Physiological Society.