Figure 4. VEGFR1 is palmitoylated in endothelial cells to regulate turnover.

(a) Workflow for acyl-resin assisted capture (Acyl-RAC). PA, palmitic acid; MMTS, methyl-methanethiosulfonate. (b) Acyl-RAC and immunoblot of HUVEC with indicated treatments. 2-BH, 2-bromohexadecadnoic acid; three replicates. (c) Acyl-RAC and immunoblot of VEGFR1 from HUVEC with indicated treatments for 45 min. CHQ, chloroquine; three replicates. (d) Diagram illustrating palmitic acid addition and removal. DHHC, protein acetyl-transferase; APT, acyl-protein thioesterase. (e,f) (e) VEGFR1 immunofluorescence of HUVEC with indicated treatments for 24 h, scale bar: 25 μm. (f) Quantification of fluorescence via integrated density. Shown are means+95% CI. (No. of cells: ntRNA, n=21; siDHHC3, n=22); four replicates. (g) Immunoblot of mVEGFR1 from HUVEC with indicated treatments; three replicates. (h) Acyl-RAC and immunoblot of HUVEC with indicated treatments for 24 h; three replicates. (i) Immunoblot of VEGFR1 and HA-tagged DHHC3 in HUVEC with indicated treatments for 24 h; three replicates. (j,k) Immunoblot of HUVEC with indicated treatments at 48 h post knockdown and treated with 50 ng ml⁻¹ VEGF-A (j) or PLGF (k); three replicates. (l–n) (l) HUVEC angiogenic sprouts with indicated treatments, scale bar: 100 μm. (m,n) Quantification of angiogenic parameters. (sprouts per bead no. of beads: ntRNA, n=25; siDHHC3, n=27; sprout length, no. of sprouts: ntRNA, n=288; siDHHC3, n=365); two replicates. Statistics: Students t-test and post-hoc Tukey's range test. **P≤0.01.