Fig. 4
The effects of Cav-3 mutations at SUMO consensus sites on Nav1.5 abundance in transfected HEK293 cells. a Schematic showing Cav-3 SUMO consensus sites. Cav-3 has a SUMO consensus motif (ΨKxD/E) near the caveolin scaffolding domain at the N-terminus. The SUMO consensus sites K38 and K144 at the C-terminus are highly conserved across different species. b-c Changes in Nav1.5 abundance in transfected HEK293 cells between the WT and mutants. HEK293 cells were co-transfected with SUMO machinery, including HA-SUMO2/3, ubc9, Flag-hPIASy, Nav1.5, and Flag-tagged Cav-3 (WT, K38R mutant or K144R mutant). Membrane protein was assessed by Western blotting at 48 h after transfection. Immunofluorescence staining was performed with an anti-Nav1.5 antibody (red, Nav1.5, arrows) and DAPI (blue, nucleus). d–e Effects of Cav-3 mutations on Nav1.5 abundance after H/R. HEK293 cells transfected with SUMO machinery, Nav1.5, and Cav-3 (WT, K38R mutant or K144R mutant) were exposed to 3 h hypoxia followed by 2 h reoxygenation. Membrane and cytoplasmic proteins were immunoblotted using anti-Nav1.5 and anti-flag (Cav-3) antibodies, respectively. Na+/K+ ATPase and β-actin were used as internal controls for membrane and cytoplasmic proteins, respectively. Cav-3 caveolin-3, DAPI 4',6-diamidino-2-phenylindole, HEK293T human embryonic kidney 293 T cells, H/R hypoxia/reoxygenation, Na+/K+ ATPase anti-sodium potassium ATPase, Nav1.5 voltage-gated sodium channel 1.5, SUMO small ubiquitin-related modifier, Ubc9 ubiquitin-conjugating enzyme 9, WT wild-type