SHKBP1 interacts with p62 under resting conditions and upon blockade of neddylation and proteasomal degradation. (A) Western blot analysis of whole-cell lysates (WCL) and α-GFP immunoprecipitates from HeLa cells cotransfected with HA-p62 and GFP-SHKBP1 or GFP EV as a control. (B) Western blot analysis of WCL and α-GFP IP from HeLa cells cotransfected with GFP-p62 and HA-SHKBP1 or HA EV as a control. (C and D) Western blot analysis of WCL and α-GFP IP to assess the interaction between SHKBP1 and exogenous (C) or endogenous p62 (D). HeLa cells were transfected with GFP-SHKBP1 in combination with HA-p62 (C) or alone (D), and treated with DMSO or the proteasome inhibitor MG-132 (20 μM) for 2 h or MLN4924 (10 μM) for 16 h. (E) HeLa cells were cotransfected with GFP-p62 and mScarlet-i-SHKBP1, treated with DMSO or the proteasome inhibitor MG-132 (20 μM) for 2 h or MLN4924 (10 μM) for 16 h, and then observed under confocal microscope 24 h after transfection. Scale bars: 10 μm. EV, empty vector. Source data are available for this figure: SourceData F2.