Fig. 3

G6PD interacts with VDAC1 under PDGF-BB stimulation. A, B Endogenous VDAC1–G6PD interactions in VSMCs detected by coimmunoprecipitation (IP) experiments. VSMCs were left unstimulated or stimulated with PDGF-BB for 12 h. Cell lysates were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies. C, D HEK293A cells were cotransfected with the Flag-VDAC1 plasmid and the HA-G6PD plasmid. The cell lysates were precipitated with an anti-HA or anti-Flag antibody, and the precipitates were analyzed by immunoblotting with an anti-Flag or anti-HA antibody, respectively. E GST pulldown analysis using recombinant glutathione S-transferase (GST)-G6PD and Flag-tagged VDAC1 (Flag-VDAC1). F The colocalization of G6PD and VDAC1 in the mitochondria of VSMCs stimulated with or without PDGF-BB. Treatment with 6AN and siG6PD was further observed. Scale bar = 10 µm. G Histogram showing the arbitrary intensities of G6PD and VDAC1 across the white arrow shown in the zoomed and merged images in F. H The Pearson correlation coefficient for signal colocalization was determined using Fiji software. I Schematic illustration of the G6PD domains used to evaluate the interaction with VDAC1. J HEK293A cells were cotransfected with the Flag-VDAC1 plasmid and the HA-G6PD-FL (FL), HA-G6PD-NTD (NTD), or HA-G6PD-CTD (CTD) plasmids. The cell lysates were immunoprecipitated with an anti-HA antibody, and the precipitates were analyzed by immunoblotting with an anti-Flag antibody. K VSMCs were transfected with HA-G6PD-FL (FL), HA-G6PD-NTD (NTD), or HA-G6PD-CTD (CTD) plasmids. The cell lysates were immunoprecipitated with an anti-HA antibody, and the precipitates were analyzed by immunoblotting with an anti-VDAC1 antibody