Fig. 4: GAL9 degrades HBc via p62-mediated selective autophagy.

a GAL9 interacts with p62. Immunoprecipitation assay was performed on HepG2 cells expressing FLAG-GAL9 and indicated HT-fused proteins. Cell lysates were precipitated with anti-FLAG antibody, followed by immunoblotting. b p62 is required for GAL9 activity. HepG2 cells were transduced with control- (Ctrl) or p62-targeting siRNA, and then transfected with vectors expressing HA-HBc and GFP-GAL9. Cells were subjected to immunoblotting analysis to detect the indicated proteins. c GAL9 promotes colocalization of p62 and HBc. Micrographs show HepG2 cells expressing FLAG-HBc (blue) with or without GFP-GAL9 (green). Endogenous p62 was stained in red. Scale bar, 10 µm. Another view of the cell image is shown in Supplementary Fig. 5b. d, e The ubiquitin-binding domain (UBA) and LC3-interacting region (LIR) of p62 are required for GAL9-mediated HBc degradation. HepG2 cells were transduced with p62-targeting siRNA, and then transfected with vectors expressing HA-HBc, GFP-GAL9, and siRNA-resistant Myc-p62 (WT, ∆UBA, or ∆LIR). Cells were subjected to immunoblotting analysis to detect the indicated proteins. Bar charts in b, d, e indicate the ratio of HBc over Vinculin, as determined by densitometry, and are presented as a mean ± SD (n = 3 experiments). ****P < 0.0001, two-tailed unpaired t-test. Immunoblots and micrographs are representative of experiments with similar results (n ≥ 2). Source data are provided as a Source Data file.