Fig. 3
Effects of PIASy silencing on Cav-3 binding to SUMO2/3 and Nav1.5, and Nav1.5 distribution. a SUMO-modified Cav-3 in the rat heart. Rat myocardial lysates were collected and immunoprecipitated with an anti-Cav-3 antibody, then immunoblotting with anti-SUMO2/3 (left panel) or anti-SUMO1 antibodies (right panel) after I/R injury. b Cav-3 binding to SUMO2/3 in HEK293 cells co-transfected with Cav-3, Nav1.5, ubc9, SUMO2/3, and incremental doses of PIASy plasmid (left panel) or identical dose of PIASy plasmid and subjected to hypoxia or H/R (right panel). c Physical binding of Cav-3 and Nav1.5 in the rat heart, and Cav-3 and SUMO machinery in co-transfected HEK293 cells. Protein samples from normal left ventricle or HEK293 cells 48 h after transfection were immunoprecipitated with an anti-Cav-3 antibody and immunoblotted with an anti-Nav1.5 antibody. d Effects of PIASy shRNA on binding between Cav-3 and Nav1.5 by Co-IP in the I/R rat heart. e Cav-3 and Nav1.5 co-localization in rat myocardium. Immunofluorescence staining of the peri-infarct zone in the rat left ventricle showing the localization and expression levels of Nav1.5 (red) and Cav-3 (green), mainly on lateral membrane (arrows) and intercalated disc (arrowheads) in cardiomyocytes. Co-localization is shown in merged images. Cell nuclei were counterstained by DAPI. Scale bar = 40 μm. Cav-3 caveolin-3, Con control, Co-IP co-immunoprecipitation, DAPI 4ʹ,6-diamidino-2-phenylindole, H hypoxia, H/R hypoxia/reoxygenation, I/R ischemia/reperfusion, Nav1.5 voltage-gated sodium channel 1.5, PIASy protein inhibitor of activated STAT Y, shRNA short hairpin RNA, SUMO small ubiquitin-related modifier