Figure 3.

HRI regulates BAG3 and HSPB8, two key proteins of chaperone-assisted selective autophagy, during proteotoxic stress.A–B, cell extracts from SC and shHRI HEK293T cells treated with DMSO (control), 10 μM MG132, and 10 nM bafilomycin for 4 h were collected and either Western blot analysis was performed using antibodies probing for BAG3, HRI, HSPB8, and GAPDH (loading control) (A) or qRT-PCR was carried out for indicated genes (B). C, scrambled (SC) sequence and HRI (shHRI) HeLa cells treated either with DMSO (control) or with 5 μM MG132 for 4 h was followed by fixation with 4% paraformaldehyde for 10 min and then were subjected to immunofluorescence analysis using anti-BAG3 and anti-p62 antibodies. Enlarged panel of MG132-treated shHRI HeLa cells on the right indicates perinuclear location of BAG3-p62+ complexes. A–B and C are representative of four and three independent experiments, respectively. The scale bar represents 10 μm.