WD domain of SHKBP1 interacts with the PB1 domain of p62. (A) Domain map of SHKBP1 and truncations used in this paper. (B) Representative live-cell images showing localization of full-length (FL) and truncated forms of SHKBP1. HeLa cells were transfected with the indicated GFP-tagged SHKBP1 construct and observed by confocal microscopy 24 h after transfection. Scale bars: 10 μm. (C) Western blot analysis of α-GFP IP of lysates from HeLa cells cotransfected with HA-CUL3 and either the indicated GFP-SHKBP1 truncation or GFP EV as a control. (D) Western blot analysis of α-GFP IP of lysates from HeLa cells cotransfected with HA-p62 and either the indicated GFP-SHKBP1 truncation or GFP EV as a control. (E) Domain map of p62 and truncations used in this paper. (F) Representative live-cell images showing localization of p62 truncations. HeLa cells were transfected with the indicated GFP-tagged p62 construct and observed by confocal microscopy 24 h after transfection. Scale bars: 10 μm. (G) Western blot analysis of α-GFP IP of lysates from HeLa cells cotransfected with HA-SHKBP1 and the indicated GFP-p62 construct. (H) AlphaFold 3 structural prediction of the interaction between SHKBP1-WD domain (magenta) and p62 (green). Electrostatic interactions (blue) indicated between p62 residues K7, R18, D92, D93, and R96 and SHKBP1 residues R357, K359, D360, D362, and E367 (pink), respectively. ipTM = 0.73, pTM = 0.54. (I) Western blot analysis of α-GFP IP of lysates from HeLa cells cotransfected with the indicated FLAG-SHKBP1 and GFP-p62 constructs. (J) Quantification of FLAG-SHKBP1 from GFP IP samples to assess the strength of interaction between p62 and SHKBP1, with band intensities normalized to FLAG-SHKBP1 levels in whole-cell lysate (WCL) and to GFP-p62 levels in IP (n = 3). Exact P values (****P < 0.0001) indicated from two-way ANOVA with Tukey’s post hoc test. (K) Representative live-cell images showing localization of p62 point mutants. HeLa cells were transfected with the indicated GFP-tagged p62 construct and observed by confocal microscopy 24 h after transfection. Scale bars: 10 μm. EV, empty vector; FL, full-length. Source data are available for this figure: SourceData F3.