Cx43 is post-translationally modified by SUMO-1, -2, and -3.
A, HeLa cells were transfected with Cx43 alone or in combination with HA-SUMO-1 and HA-Ubc9. Cell lysates were subjected to immunoprecipitation (IP) using anti-Cx43 antibodies or preimmune serum (PI) as negative control. SUMOylated Cx43 was detected by Western blotting (WB) using anti-HA antibodies. The blot was stripped and reprobed using anti-Cx43 antibodies. B, as in A, but HA-SUMO-1 was replaced with HA-SUMO-3. C, HeLa cells were cotransfected with Cx43 and HA-SUMO-1, HA-SUMO-2, or HA-SUMO-3 with or without HA-Ubc9. Cell lysates were subjected to immunoprecipitation using anti-Cx43 antibodies. SUMOylated Cx43 was detected by Western blotting using anti-HA antibodies. The blot was stripped and reprobed using anti-Cx43 antibodies. D, HeLa cells were cotransfected with Cx43 or empty vector together with HA-SUMO-1, HA-SUMO-2, or HA-SUMO-3 and HA-Ubc9. Cell lysates were subjected to immunoprecipitation using an antibody against Cx43. SUMOylated Cx43 was detected by Western blotting using anti-HA antibodies. The blot was stripped and reprobed using anti-Cx43 antibodies. Molecular mass in kDa is indicated.