Fig 1: GLUT1 and glycolytic enzymes localize to PDGFR endocytic vesicles.(A) Serum-starved Swiss 3T3 fibroblasts were stimulated with PDGF-BB (50 ng/ml) for 10 min or left unstimulated (control), and then immunostained with anti-GLUT1 (green) and anti-PDGFRα (magenta) antibodies. Nuclei were stained with DAPI (blue). Higher magnification images of the boxed regions are shown on the right. Representative images are shown for each condition from one of 2 independent experiments. (B) Cell surface GLUT1 in serum-starved Swiss 3T3 cells was labeled with recombinant GLUT1-binding region of human T cell leukemia virus envelope glycoprotein for 20 min on ice, and then the cells were stimulated with PDGF-BB (50 ng/ml) for 10 min at 37°C or left unstimulated and immunostained for surface and internalized GLUT1 (green) and PDGFRα (magenta). Nuclei were stained with DAPI (blue). Higher magnification images of the boxed regions are shown on the right. Representative images are shown for each condition from one of 2 independent experiments. (C) Serum-starved Swiss 3T3 fibroblasts were stimulated with PDGF-BB (50 ng/ml) for 10 min or left unstimulated, and then subjected to PLA with anti-PDGFRβ and anti-HK1 or -HK2 antibodies (gray). Nuclei were stained with DAPI (blue). PLA signals in the indicated number of cells were counted and plotted in the graphs. Bars represent mean ± SD of PLA signals per cell. P values were calculated using two-tailed Welch’s t test. Representative data are shown from one of 2 independent experiments. (D) Serum-starved and PDGF-BB-stimulated or unstimulated cells (as above) were immunostained with anti-GPI or anti-TPI (green) and anti-PDGFRα (magenta) antibodies. Higher magnification images of the boxed regions are shown on the right. Nuclei were stained with DAPI (blue). Representative images are shown for each condition from one of 2 independent experiments. (E) Serum-starved and PDGF-BB stimulated or unstimulated cells were subjected to PLA with anti-PDGFRβ and the indicated antibodies. PLA signals were counted and plotted in the graphs. Bars represent mean ± SD of PLA signals per cell. P values were calculated using two-tailed Welch’s t test. Representative data are shown from one of 2 independent experiments. Scale bars: 20 μm.