Fig. 6: Identification of the ubiquitination site of TNC.

a Full-length Flag-TNC and TNC truncation mutants were co-transfected with HA-Ub-WT into HEK293T cells, then followed by EBSS treatment in the presence of BafA1 for 2 h. Immunoprecipitation analysis of exogenous TNC ubiquitination with the indicated antibodies. b Predict the ubiquitination lysine sites of TNC using Ubpred website (www.ubpred.org). c HEK293T cells transfected with Flag-TNC-P2 and indicated TNC mutants along with HA-Ub, then followed by EBSS treatment in the presence of BafA1 for 2 h. Immunoprecipitation analysis of exogenous TNC ubiquitination. d Coimmunoprecipitation and immunoassay of extracts of HEK293T cells transfected with the indicated plasmids. P62 proteins were immunoprecipitated and the bound exogenous TNC proteins examined by immunoblot. e HEK293T cells were transfected with the indicated plasmids, then treated with EBSS at indicated intervals and analyzed by immunoblot. All data are representative of three independent experiments.