Extended Data Fig. 8: VEGF promotes internalization of CTR1 and VEGFR2 from cell surface in a dynamin- and VEGFR2-dependent manner but CuCl2 promotes CTR1 internalization not VEGFR2.
From: Cysteine oxidation of copper transporter CTR1 drives VEGFR2 signalling and angiogenesis
A, B, C. HUVECs stimulated with VEGF (20 ng/ml) for indicated times (A) or incubated with dynasore, a dynamin-associated endocytic inhibitor (200 nM) (B) or transfected with control or VEGFR2 siRNAs were stimulated with VEGF (20 ng/ml) for 30 min (C). Cells were labeled with cell surface biotinylation reagent, 1 mM EZ-Link Sulfo-NHS-LC-Biotin, followed by wash and then lysates were pulled down with streptavidin beads, followed by IB with antibodies indicated to detect cell surface CTR1 or VEGFR2 or Na,K-ATPase (cell surface marker). D. HUVECs stimulated with CuCl2 (25 µM) for indicated times were labeled with cell surface biotinylation reagent, 1 mM EZ-Link Sulfo-NHS-LC-Biotin. After wash, lysates were pulled down with streptavidin beads, followed by IB with antibodies indicated to detect cell surface CTR1 or VEGFR2 or Na,K-ATPase. Bottom panels represent the averaged cell surface CTR1 and VEGFR2 levels expressed as fold changes from the basal control. (n = 3 biologically independent experiments). A, B and D two-tailed unpaired t-test. C, one-way ANOVA followed by Tukey’s multiple comparisons analysis, *p < 0.05, **p < 0.01, ***p < 0.001. NS = not significant. Data are mean ± SEM). Source numerical data and unprocessed blots are available in source data.
