Figure 5: TRB3 interacts with p62 disturbing the p62 cargo functions.

(a) HEK 293T cells were co-transfected with the expression plasmids of p62-DDK and TRB3-HA. Cell extracts were IP with anti-DDK Ab and blotted with anti-HA Ab. Data are representative immunoblots of five independent assays. (b) HepG2 extracts were IP with anti-p62 Ab or rabbit IgG and were blotted with anti-TRB3 Ab. Data are representative immunoblots of five independent assays. (c) In vitro interaction of TRB3/p62 was detected with GST pull-down assay. Retrieved proteins were examined by immunoblotting. GST-only protein was used as negative control. GST fusion proteins were stained with Coomassie Blue. Data are representative immunoblots of three independent assays. (d) Colocalization of TRB3 and p62 was detected by immunostaining. Data are representative images of three independent assays. Scale bar, 10 μm. (e) Mapping of p62 regions involved in TRB3 binding. Top: deletion mutants of p62. Below: HEK 293T cells were co-transfected with indicated constructs of p62 and TRB3-HA. Cell extracts were IP with anti-HA Ab. Data are representative immunoblots of three independent assays. (f) HEK 293T cells were transiently transfected with indicated plasmids. The p62-LC3 binding was evaluated with IP assay. Data are representative immunoblots of three independent assays. (g) HEK 293T cells were transfected with Ub-DDK plus or minus TRB3-HA plasmids. The binding of p62-ubiquitinated protein was evaluated with IP assay. Data are representative immunoblots of three independent assays. (h,i) The colocalization of P62/LC3 (h) or p62/ubiquitinated proteins (i) was detected by immunostaining. Scale bar, 7.5 μm. Data are representatives of three assays.