CipB is primarily ubiquitinated at lysine 218 (K218) through K27-linked polyubiquitin chains. (A) Denature-IP showing the binding of Flag-CipB to HA-tagged ubiquitin variants in 293T cells. Cells were lysed and subjected to denature-IP. The immunoprecipitate was eluted with FLAG peptide and analyzed by immunoblotting. (B) Denature-IP analysis of HA-CipB and Flag-tagged ubiquitin variants. Cells were lysed and subjected to denature-IP. The immunoprecipitate was eluted with SDS loading buffer and analyzed by immunoblotting. (C) Confocal microscopy analysis of the colocalization between RFP-CipB (magenta) and GFP-ubiquitin lysine variants (cyan). Scale bar, 10 μm. (D) Quantification of Pearson’s colocalization coefficient between RFP-CipB and ubiquitin lysine variants. Mean ± SEM, n = 25 cells in each group. P values were determined by one-way ANOVA with Tukey’s test. ns, not significant; ***P < 0.01. (E) Denature-IP showing the binding of HA-ubiquitin (K27O) to Flag-CipB mutations in 293T cells. Cells were lysed and subjected to denature-IP. The immunoprecipitate was eluted with FLAG peptide and analyzed by immunoblotting. (F) CipB induces p62 recruitment to mitochondria in HeLa cells. Cells were co-transfected with Mito-GFP (magenta) and RFP-tagged (blue) plasmids for 14 h, followed by immunostaining for endogenous p62 (cyan). Positive control: cells co-transfected with RFP-Parkin (blue) and Mito-GFP were treated with 10 μM CCCP for 6 h to induce p62-mitochondria colocalization. Scale bar: 10 μm. All data are representative of three independent experiments. Source data are available for this figure: SourceData FS3. IP, immunoprecipitation.