SHKBP1 inhibits p62 oligomerization without affecting its ubiquitination state. (A) Western blot analysis of in vivo ubiquitination assay. WT or SHKBP1 KO HeLa cells were cotransfected with His-ubiquitin and either GFP-p62 or GFP EV, and OE group represents WT cells that were also cotransfected with FLAG-SHKBP1. 24 h after transfection, cells were treated with MG-132 (20 μM) for 2 h and subjected to His pull-down using Ni-NTA agarose and western blot. (B) Western blot analysis of whole-cell lysates (WCL) from WT, SHKBP1 KO, and FLAG-SHKBP1–OE HeLa cells. (C) Representative live-cell images showing p62 body formation. WT or SHKBP1 KO HeLa cells were transfected with GFP-p62 either alone (left) or in combination with mScarlet-i-SHKBP1 (right, where OE refers to SHKBP1 OE in WT cells, and RE (rescue) refers to SHKBP1 OE in KO cells) and imaged using confocal microscopy 24 h after transfection. Scale bars: 20 μm. (D) Quantification of the average p62 body size of images shown in (C) from three independently plated samples. n = 51 (KO), 43 (WT), 37 (OE), and 35 (RE). Exact P values indicated (****P < 0.0001) from one-way ANOVA with Tukey’s post hoc test. (E) Western blot analysis of WCL from WT or SHKBP1 KO HeLa cells after DSP cross-linking. Cells were treated with MG-132 (0.5 μM) for 12 h, cross-linked with 0.4 mg/ml DSP at 4°C for 2 h, and lysed in IP lysis buffer with 1% SDS. The lysates were mixed with reducing or nonreducing loading buffer (i.e., with or without β-mercaptoethanol) and were analyzed by western blot. (F) Quantification of the ratio of intensities of monomeric to total p62. Intensities were normalized to the WT without the MG-132 treatment group. n = 6. Exact P values indicated (****P < 0.0001) from two-way ANOVA. EV, empty vector.