FIGURE 5.

Identification of Lys-144 and Lys-237 as Cx43 SUMOylation sites. HeLa cells were transfected with Cx43-WT (A, B), Cx43-K144R (A) or Cx43-K237R (B) in combination with HA-SUMO-1, -2, or -3 and HA-Ubc9, as indicated. Cell lysates were subjected to immunoprecipitation (IP) using anti-Cx43 antibodies. SUMOylated Cx43 was detected by Western blotting (WB) using anti-HA antibodies. The blots were stripped and reprobed using anti-Cx43 antibodies. The intensities of the HA signals were measured and normalized to the intensities of the Cx43 signals in the same samples. The SUMOylation level for Cx43-K144R and Cx43-K237R is expressed in percentage relative to Cx43-WT. Values shown are the mean ± S.D. of three independent experiments. Molecular mass in kDa is indicated. C, schematic overview of the location of Lys-144 and Lys-237 and other lysine residues in Cx43. Blue indicates that the lysine acts as a SUMO conjugation site. D, amino acid sequence of the human Cx43 regions that contain Lys-144 and Lys-237 and corresponding sequences in the indicated species homologs (upper panel) or in other connexin isoforms in humans (lower panel).