WDR81 interacts with LC3C through two LIRs to facilitate aggrephagy. (A and B) Co-IP of GFP-tagged WDR81 fragments with mCh-LC3C. Individual GFP-WDR81 fragments were coexpressed with mCh-LC3C in HEK293 cells. IPs were performed with mCherry antibody, and precipitated proteins were detected with antibodies to GFP and mCherry. (C) Alignment of canonical and noncanonical LIRs in WDR81 with those in p62, NBR1, FUNDC1, or NDP52. Conserved residues are shown in red. (D) Co-IP of mCh-LC3C with wild-type (WT), LIR, or CLIR mutants of GFP-WDR81(341–650) in HEK293 cells. WDR81 mutations are shown in the box. IPs were performed with mCherry antibody and precipitated proteins were detected with antibodies to GFP and mCherry. (E) Co-IP of mCh-LC3C with WT or the indicated LIR mutants of Flag-WDR81 full-length protein in HEK293 cells. IPs were performed as in A. (F) Pull-down of Flag-WDR81 by GST-LC3C. Purified recombinant GST and GST-LC3C immobilized on glutathione-Sepharose beads were incubated overnight at 4°C with lysates of HEK293 cells expressing Flag-WDR81 or the indicated Flag-WDR81 mutants. After extensive washing, bound proteins were subjected to Western blot with Flag antibody. (G and H) KO-81 HeLa cells were transfected with Flag-WDR81 or the indicated Flag-WDR81 mutants. 48 h later, cells were subjected to immunostaining (G) or immunoblotting (H) with p62 antibody. Quantification (mean ± SEM) of p62 in (G) is shown in the right panel. ***, P < 0.001. Bars, 10 µm. (I) Co-IP of Flag-WDR81 with WT or the indicated hydrophobic pocket mutants of LC3C in HEK293 cells. IPs were performed with Flag antibody, and precipitated proteins were detected with antibodies to Flag and mCherry. (J) Immunoblotting of p62 in siCtrl or siLC3C cells expressing RNAi-resistant LC3C (WT) or the LC3C (K32Q/F33H/L64V/F69) mutant in HEK293 cells. In H and J, fold changes in p62 levels were normalized to the control and are indicated at the bottom.