Fig. 4

Endoplasmic Hrd1 selectively sequesters mutant Blimp-1 proteins, and promotes their ubiquitination and cytoplasmic degradation. a HeLa cells co-expressing mCherry-Hrd1 with GFP-tagged WT or mutant Blimp-1 proteins were treated with MG132 for 24 h and observed under a confocal microscope. Scale bar: 10 μm. b Hrd1 ^−/− 293T cells expressing GFP-tagged WT or mutant Blimp-1 proteins were treated with MG132 for 12 h and observed under a confocal microscope. Scale bar: 7.5 μm. c Hrd1 ^−/− 293T cells co-expressing mCherry-Hrd1 with GFP-tagged WT or mutant Blimp-1 proteins were treated with MG132 for 12 h and observed under a confocal microscope. Scale bar: 7.5 μm. d Flag-tagged WT or mutant Blimp-1 proteins were overexpressed in 293T cells, which were then treated with MG132 (20 μM) or left untreated for 12 h. The extracts were co-immunoprecipitated with control IgG or an anti-flag antibody (upper panel)/Hrd1 antibody (bottom panel). The co-immunoprecipitated lysates and extract input were then immunoblotted with antibodies against the indicated proteins. e Flag-tagged WT or mutant Blimp-1 proteins were overexpressed in 293T cells, Hrd1 ^−/− 293T cells or Hrd1 ^−/− 293T cells expressing mCherry-Hrd1. The cells were treated with MG132 for 24 h before extracts were immunoprecipitated with an anti-flag antibody and immunoblotted with an anti-Ub antibody