Fig. 1.

The SUMOylated AnxA6 is detected under condition of protein overexpression. A-B The SUMOylated AnxA6 is mainly modified by SUMO1 in HEK293T cells. HEK293T cells were transiently co-transfected with pFlag-AnxA6, pHA-UBC9 and pHis-SUMO1(SUMO2/SUMO3) plasmids as indicated. Cells were collected at 48 h after transfection, and target proteins were captured by IP on anti-Flag antibody coupled agarose beads (A) or Ni²⁺-NTA agarose beads (B). Then the target protein was detected by immunoblotting with anti-Flag and anti-His antibodies. C-D The SUMOylation of AnxA6 was detected in response with UBC9/SUMO1 enhancement or inhibitor treatment (2-D08 or ML792). HEK293T cells were co-transiently transfected with pFlag-AnxA6 and pHA-UBC9 or pHis-SUMO1 plasmids as indicated. After transfection for 24 h, cells were treated with 150 μM 2-D08 (C) or 10 μM ML792 (D) for another 24 h. The Flag-tagging AnxA6 protein was enriched by IP on anti-Flag antibody coupled agarose beads (C) or Ni²⁺-NTA agarose beads (D), from which the SUMOylated AnxA6 level was analyzed through western blot using Flag or SUMO1 antibody. S-Flag-AnxA6: SUMOylated Flag-tagging AnxA6, IP: immunoprecipitation, IB: immunoblot, Input: Same account of cell lysate to load. 2-D08 or ML792: SUMOylation inhibitor