Fig. 2

Collagen IV follows a post-Golgi secretory pathway. (a) Representative images of ECs treated with brefeldin A (BFA) at indicated time points after BFA exposure. Cells were stained for collagen IV (Col IV) (red), actin (green), and DNA (blue). Arrowheads denote extracellular Col IV secretion. (b) Graph of Col IV extracellular ratio in control or BFA-treated ECs at indicated time points after BFA exposure. N number of cells. (c) Immunoblot of Col IV in ECs treated with cycloheximide (CHX) (20 mg/ml) and probed for indicated proteins. (d) Immunoblot of Col IV in ECs treated with both CHX + BFA and probed for indicated proteins. (e) Graph of Col IV protein accumulation in ECs treated with CHX, and both CHX + BFA. Col IV accumulations were normalized to α-tubulin levels. N number of replicates. (f) Immunoblot of Col IV in ECs transfected with either scramble or Rab10 siRNA and treated with CHX. EC lysate probed for indicated proteins. (g) Graph of Col IV levels in ECs transfected with either scramble or Rab10 siRNA and treated with CHX. Col IV accumulations were normalized to α-tubulin levels. N number of replicates. (h) Representative images of ECs cultured in VEGF-containing or serum-starve (SS) media and stained for Col IV (red), actin (green), and DNA (blue). Dotted green line indicates cell outline. Arrowheads denote extracellular Col IV secretion. (i) Graph of Col IV extracellular ratio of ECs cultured in VEGF-containing or SS media. N number of cells. (j) Immunoblot of Col IV in ECs cultured in VEGF-containing or SS media treated with CHX for the indicated time and probed for indicated proteins. (k) Immunoblot of Col IV in ECs cultured in VEGF-containing or SS media treated with CHX and chloroquine (CLQ) (10 µM) for the indicated times and probed for indicated proteins. (l) Immunoblot of Col IV in ECs transfected with either scramble or Rab10 siRNA and cultured in VEGF-containing or SS media treated with CHX and/or BFA for 8hrs and probed for indicated proteins. (m) Immunoblot of Col IV in Human umbilical vein ECs (HUVEC), Human aortic ECs (HAEC), Human brain microvascular ECs (HBMEC) and Human dermal ECs (HDEC) cultured in either VEGF-containing or SS media and probed for indicated proteins. For all experiments, data represented as mean ± 95% confidence intervals. Black bars indicate comparison groups with indicated p-values. All p-values are from two-tailed Student’s t-test from at least three experiments. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001; ns not significant