Fig. 5

AnxA6 SUMOylation is identified to conjugate with SUMO1 at K579 residue, and SENP1 cooperates with AnxA6 to perform deSUMOylation of AnxA6 in HCC cells. (A-B) K579 site of the peptide ⁵⁶⁹MTNYDVEHTIKK⁵⁸⁰ from AnxA6 was identified to SUMOylate by MS/MS analysis in HCD fragmentation mode. (C) The K579 site mutation reduced AnxA6 SUMOylation level in HepG2 cells. pFlag-AnxA6^K75R, pFlag-AnxA6^K306R, pFlag-AnxA6^K418R and pFlag-AnxA6^K579R were AnxA6 site mutated plasmids, each with a single K point mutation, to validate SUMOylation level changes of AnxA6 by western blot (left). Quantification of the SUMOylated AnxA6 levels in HepG2 cells (right). The bar chart represented the ratio of SUMOylated Flag-tag AnxA6 to the total AnxA6 protein in the IP elution solution. ns, no statistical; *P < 0.05; **P < 0.01; ***P < 0.001. (D-E) SENP1 cooperated with AnxA6 to perform AnxA6 deSUMOylation. HepG2 cells were transfected plasmids pFlag-AnxA6 with or without pFlag-SENP1 for 48 h to detect by IP using the anti-Flag antibody. (F) SENP1 overexpression decreased the SUMOylation of AnxA6 in HepG2 cells. HepG2 cells were transfected pFlag-AnxA6 with or without pFlag-SENP1 for 48 h to detect by IP using the anti-Flag antibody (left). Quantification of the SUMOylated AnxA6 levels in HepG2 cells transfected pFlag-AnxA6 with or without pFlag-SENP1(right). The bar chart represented the ratio of SUMOylated Flag-tag AnxA6 to the total AnxA6 protein in the IP elution solution. ****P < 0.0001. S-AnxA6: Flag-tagging SUMOylated AnxA6, IP: immunoprecipitation, IB: immunoblot, Input: same account of cell lysate to load