Fig. 2
From: SUMOylation of AnxA6 facilitates EGFR-PKCα complex formation to suppress epithelial cancer growth
K299 residue is one main SUMOylation site of AnxA6. A The SUMOylated AnxA6 was enriched and detected by His-SUMO1T95K tag. The His-SUMO1T95K-AnxA6 conjugated protein was enriched by IP on anti-Flag antibody coupled agarose beads, then the target protein was detected by immunoblotting with anti-Flag and anti-His antibodies. B-C MS/MS spectra of AnxA6 peptide DAYERDLEADIIGDTSGHFQK156, YEK299SLYSMIK, NDTSGEYK314K in HCD fragmentation mode. And the K156, K299, K314 were identified as the SUMOylation sites. D Several lysine residues of AnxA6 were predicted to be potential the consensus SUMOylation sites by a bioinformatics software analysis. E–G Site mutant K299R significantly reduced SUMOylation level of AnxA6. HEK293T cells were co-transfected with plasmids pFlag-AnxA6, each of the single mutants, or pHis-SUMO1 as indicated. After 48 h later, cell lysates were performed IP to capture Flag-tagging AnxA6 (E) or His-tagging SUMO1 (F), which was immunoblotted with anti-SUMO1 antibody to show SUMOylation level. G HEK293T cells were transfected with pFlag-AnxA6 or pFlag-AnxA6K299R plasmids along with pHis-SUMO1 to measure AnxA6 SUMOylation level. The levels of SUMOylation were determined as described in Methods. S-Flag-AnxA6: SUMOylated Flag-tagging AnxA6, IP: immunoprecipitation, IB: immunoblot, Input: same account of cell lysate to load