Fig. 1

ACTL6A interacts with and stabilizes MYC protein via abrogating GSK3β-induced MYC degradation. a Lysates from SUM159PT cells were immunoprecipitated using an anti-MYC antibody, followed by MS peptide sequencing. ACTL6A was identified in the precipitate. b Representative MS plots and sequences of peptides from ACTL6A. c Reciprocal immunoprecipitation (IP) assay revealed the interaction between MYC and ACTL6A in SUM159PT and 293FT cells. d Western blot analysis of ACTL6A and MYC protein in control, ACTL6A-overexpressed and -knockdown SUM159PT cells. GAPDH was used as loading control. e Western blot analysis of MYC protein in the indicated cells treated with CHX (50 μg/mL) for 0, 30, 60, or 120 min. GAPDH was used as loading control. f MYC protein level in the indicated cells under MG132 (10 μM) treatment for 8 h and then western blotting was conducted. GAPDH was used as loading control. g Effect of ubiquitination on MYC by immunoprecipitation assay in SUM159PT cells. HA-tagged ubiquitin, ACTL6A-overexpressed plasmids, and ACTL6A shRNA plasmids were co-transfected into SUM159PT cells with Flag-tagged MYC plasmid. Immunoprecipitated proteins with anti-HA antibody were detected by MYC antibody and whole cell lysates were immunoblotted as loading controls. h The truncations of MYC and ACTL6A were based on known functional domain. i, j Detailed interactions between MYC and ACTL6A were analyzed by IP assays. k IP assay showed that overexpressing ACTL6A substantially decreased the interaction between MYC and GSK3β. l Transfected with wild-type MYC and different mutant MYC into SUM159PT cells and were analyzed by western blotting to detect the phosphorylated levels of MYC