Fig. 4: ATP7A knockdown enhances lysosomal degradation of VEGFR2.
a Human umbilical vein endothelial cells (HUVECs) transfected with control or ATP7A siRNAs were stimulated with vascular endothelial growth factor (VEGF) (20 ng/ml). Lysates were used for cell surface biotinylation using sulfo-NHS-SS-biotin to measure cell surface VEGFR2 or ATP7A by biotin pull-down, followed by immunoblotting (IB) with anti-VEGFR2 or ATP7A antibody (Ab). Non-immunoprecipitated (IP) total cell lysate was used for IB with Abs indicated. The bar graph represents the averaged cell-surface VEGFR2 or ATP7A protein levels over the basal control. n = 3, VEGFR2: **p = 0.0066, **p = 0.0046; ATP7A: *p = 0.047, *p = 0.0343 (two-tailed unpaired t-test). b HUVECs transfected with control or ATP7A siRNAs stimulated with VEGF (20 ng/ml) for 15 min were used for co-localization analysis for VEGFR2 and Rab5 or Lamp2. Yellow fluorescence in merged images indicates their colocalization. Scale bars = 10 μm. The bar graph represents the percentage of colocalization with VEGFR2. n = 8, Rab5: #p < 0.0001; lamp2: #p < 0.0001 (two-tailed unpaired t-test). c HUVECs transfected with control or ATP7A siRNAs and stimulated with VEGF (20 ng/ml) for 30 min were used for co-localization analysis for VEGFR2 and Rab7 using the corresponding antibodies. For VEGFR2-Rab11 colocalization, Rab11 was detected through Rab11-GFP plasmid over-expression and VEGFR2 was detected through an anti-VEGFR2 antibody. Yellow fluorescence in merged images indicates their colocalization. Scale bars = 10 μm. The bar graph represents the percentage of colocalization with VEGFR2. n = 4, Rab7: **p = 0.009; Rab11: *p = 0.0128 (two-tailed unpaired t-test). d, e HUVECs transfected with control or ATP7A siRNAs were pretreated with lysosome inhibitor chloroquine (500 µM for 30 min) or proteasome inhibitors MG132 (carbobenzoxy-Leu-Leu-leucinal) (10 µM for 30 min) for 30 min, or lactacystin (10 µM for 60 min) (d) or bafilomycin A1 (5 nM for 60 min) (e) followed by VEGF stimulation for 30 min. Lysates were IB with anti-VEGFR2 or actin loading control. d n = 3, **p = 0.009; e n = 3, *p = 0.0223, **p = 0.0027, *p = 0.0122 (two-tailed unpaired t-test). Data are mean ± SEM.
