Fig. 5

KEAP1-259aa interacts with ARIH1 to promote vimentin degradation. (A). Total immunoprecipitated proteins from Flag-KEAP1-259aa cells were separated via SDS-PAGE. Vimentin and ARIH1 were identified by LC/LC-MS. (B). Interaction of GFP-vimentin, Myc-ARIH1 and Flag-KEAP1-259aa were determined by co-IP assay. (C). Flag-tagged KEAP1-259aa and GFP-vimentin were co-transfected into MG63 cells and immunofluorescence staining was performed using anti-Flag and anti-GFP antibodies (scale bar, 20 μm). (D). Expression of vimentin in OS cells transfected with the indicated plasmid or siRNAs following the treatment with CHX (50 µg/ml). (E). Expression of vimentin was detected in OS cells transfected with the indicated plasmid or siRNAs following the treatment with MG132 (10 µM). (F). Ubiquitin of vimentin, in MG132 (10 µM)-treated cells, was detected by IP using GFP antibodies in the indicated cells followed by immunoblotting using HA antibodies. (G). Wild-type HA-Ub, K11R-, K48R- or K63R-mutant HA-Ub plasmids were transfected into OS cells. Wild-type HA-Ub, K11 HA-Ub plasmids were transfected into OS cells. Ubiquitin of vimentin was detected by IP assay in the treatment of MG132 (10 µM). (H). Wild-type GFP-vimentin, or vimentin S39A, S56A or S73A mutant plasmids were transfected into OS cells. The binding of vimentin and KEAP1-259aa was detected by co-IP assay. (I). Wild-type GFP-Vimentin, or vimentin S39A, S56A or S73A mutant plasmids were transfected into OS cells. Ubiquitin of vimentin was detected by IP assay in the treatment of MG132 (10 µM). Error bars represent three independent experiments. *, **, *** indicates significant differences compared with the control group at a p value < 0.05, < 0.01, < 0.001, respectively