SHKBP1 inhibits Keap1 aggregation and clearance via p62 bodies. (A) Western blot analysis of co-IP experiments for endogenous p62. Lysates were from WT HeLa cells, SHKBP1 KO HeLa cells, and HeLa cells transfected with mScarlet-i-SHKBP1 (OE). (B) Quantification of Keap1 from p62-IP to demonstrate the interaction between endogenous p62 and Keap1, with band intensities normalized to actin levels (n = 3). Exact P values indicated from one-way ANOVA with Tukey’s post hoc test. (C) Western blot analysis of whole-cell lysates (WCL) from WT and SHKBP1 KO HeLa cells treated with As(III) (10 µM) for the indicated times. (D) Quantification of Keap1 protein levels, with band intensities normalized to actin levels (n = 5). Exact P values indicated (****P < 0.0001) from two-way ANOVA. (E) Confocal microscopy analysis of WT and KO SHKBP1 HeLa cells cotransfected with GFP-p62 and miRFP-Keap1 and treated with As(III) (10 µM) for 4 h or with DMSO as a control. Scale bars: 20 μm. (F and G) Quantification of Keap1 aggregate size (F) and p62 body size (G) in WT and SHKBP1 KO HeLa cells after As(III) treatment (representative images shown in 6E). n = 11 images from three independently plated samples. Exact P values indicated from unpaired two-tailed Student’s t test. (H) Confocal microscopy analysis of HeLa cells cotransfected with GFP-p62 and miRFP-Keap1, with or without mScarlet-i-SHKBP1, and treated with As(III) (10 µM) for 4 h or with DMSO as a control. Scale bars: 20 μm. (I and J) Quantification of p62 body size (I) and the percentage of cells containing Keap1 aggregates (J) in control of SHKBP1-OE HeLa cells with or without As(III) treatment (representative images shown in 6H). n = 8–12 images from three independently plated samples. Exact P values indicated from two-way ANOVA. (K) Immunofluorescence (IF) analysis of endogenous p62 and Keap1 in WT and SHKBP1 KO HeLa cells by confocal microscopy. Cells were treated with As(III) (10 µM) for indicated times. Scale bars: 10 μm; 5 μm (zoomed-in images). (L–N) Quantification of Keap1 aggregate size (L), p62 body size (M), and colocalization of Keap1 and p62 (N) in WT and SHKBP1 KO HeLa cells with As(III) treatment for the indicated times (representative images shown in 6K). n = 13–18 images from three independently plated samples. Exact P values indicated (****P < 0.0001) from two-way ANOVA with Tukey’s post hoc test. Source data are available for this figure: SourceData F6.