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. 2022 Jun 1;23(7):e53855. doi: 10.15252/embr.202153855

Figure EV5. CBX4 SUMOylates EZH2 with SUMO1, SUMO2 and SUMO4.

Figure EV5

  1. HA‐tagged EZH2 was co‐overexpressed with Flag‐tagged SUMO1, GFP‐tagged UBC9 or Flag‐tagged CBX4. EZH2 was IP with anti‐HA beads. Both total and IP samples were IB with anti‐HA, ‐Flag, and ‐GAPDH antibodies. The expression ratios of SUMO1‐EZH2 are marked below the panel.
  2. Target proteins were overexpressed, IP and IB as in (A), except that Flag‐tagged SUMO molecule was changed with SUMO2. The expression ratios of SUMO2‐EZH2 are marked below the panel.
  3. Target proteins were overexpressed, IP and IB as in (A), except that Flag‐tagged SUMO molecule was changed with SUMO4. The expression ratios of SUMO4‐EZH2 are marked below the panel.
  4. Primary CD4+ T cells from three healthy donors were transfected with siNC or siCBX4 utilizing 4D‐Nucleofector System. About 24 h post transfection, the endogenous EZH2 within each group was IP with anti‐EZH2 antibodies. Both total and IP samples were IB with anti‐EZH2, anti‐CBX4 and anti‐GAPDH antibodies. The expression ratios of SUMO‐EZH2 within each group are marked below the panel.
  5. HA‐tagged EZH2 and EZH2 truncation mutants were co‐overexpressed with Flag‐tagged SUMO4, Flag‐tagged UBC9 and Flag‐tagged CBX4. EZH2 and corresponding mutants were IP with anti‐HA beads. Both total and IP samples were IB with antibodies against HA, Flag and GAPDH. The asterisks represent SUMOylated EZH2 mutants.