Figure 2: Palmitoylation regulates mVEGFR1 stability.
From: Dynamic alterations in decoy VEGF receptor-1 stability regulate angiogenesis
(a,b) (a) VEGFR1 immunofluorescence of HUVEC with indicated treatments. (b) Quantification of fluorescence via integrated density. (No. of cells: EtOH/H2O, n=20; EtOH/CHX, n=12; 2-BH/H2O n=24; 2-BH/CHX n=18); CHX, cycloheximide; two replicates, scale bar: 25 μm. (c) Immunoblot of HUVEC with indicated treatments; four replicates. (d,e) (d) VEGFR1 immunofluorescence of d5 HUVEC angiogenic sprouts with indicated treatments; 2-BH, 2-bromohexadecadnoic acid, scale bar: 25 μm. (e) Quantification of fluorescence via integrated density. (No. of sprouts: EtOH/H2O, n=20; EtOH/CHX, n=12; 2-BH/H2O, n=24; 2-BH/CHX, n=18); two replicates. (f) Quantification of VEGFR1 fluorescence in HUVEC with indicated treatments via integrated density. (n=50 cells per condition); four replicates. (g) Immunoblot of HUVEC stimulated with 50 ng ml−1 PLGF or PBS for indicated times; four replicates. (h) Quantification of VEGFR1 fluorescence of HUVEC with indicated treatments and 50 ng ml−1 PLGF or VEGF-A via integrated density. Pal-B, palmostatin-B. (No. of cells: DMSO/PBS, n=51; DMSO/PLGF, n=37; DMSO/VEGF-A, n=45; Pal-B/PBS, n=50; Pal-B/PLGF n=48; Pal-B/VEGF-A, n=51); three replicates. (i) Immunoblot of HUVEC with indicated treatments and 50 ng ml−1 PLGF or VEGF-A for 1 h. Values are mVEGFR1 normalized to respective PBS controls; three replicates. (j,k) (j) VEGFR1 immunofluorescence of d5 HUVEC angiogenic sprouts with indicated treatments and 75 ng ml−1 PLGF or PBS for 4 h, scale bar: 25 μm. (k) Quantification of fluorescence via integrated density. (No. of sprouts: DMSO/PBS n=13; DMSO/PLGF, n=17; Pal-B/PBS n=12; Pal-B/PLGF n=16); two replicates. Statistics: Shown are means+95% CI. One-way ANOVA with pairwise comparison and post-hoc Tukey’s range test. *P≤0.05; **P≤0.01; NS, not significant.
