Cytoskeleton Disruption and Protein Synthesis Inhibition in HUVECs

Latrunculin B (Calbiochem-Novabiochem Corp., San Diego, CA, USA) and nocodazole (Sigma-Aldrich, Saint Louis, MO, USA) were used as previously described to disrupt actin and microtubule cytoskeleton integrity, respectively [27 (link)]. Cycloheximide (Sigma-Aldrich) was used to inhibit protein synthesis in HUVECs at the concentration of 50 μg/mL.
The following primary antibodies were used: mouse monoclonal anti-CD93 (mAb 4E1) [6 (link)], rabbit anti-MMRN2 (generously provided by M. Mongiat), rabbit anti-CD93 (HPA009300, Atlas Antibodies, Bromma, Sweden), mouse anti-CD93 (MBL International Corporation, Woburn, MA, USA), rabbit anti-Giantin, mouse anti-β1 integrin (12G10), and mouse anti-Rab7 (Abcam, Cambridge, UK), rabbit anti-Moesin (Cell Signaling Technology, Danvers, MA, USA), mouse anti-Rab5 (BD Biosciences, Franklin Lakes, NJ, USA), mouse anti-β-actin (Sigma-Aldrich), rabbit anti-CD93 (H-190), mouse anti-COPD (E-12), mouse anti-Sec31A (H-2), mouse anti-β-Adaptin (A-5), mouse anti-Rab5a (E-11), mouse anti-Rab5b (F-9), mouse anti-Rab5c (H-3), mouse anti-β1 integrin (4B7R), mouse anti-Rab11a (D-3), rabbit anti-caveolin-1 (N-20), and mouse anti-MMRN2 (H572) (Santa Cruz Biotechnology, Dallas, TX, USA). Alexa Fluor-488 and -647 phalloidin (Thermo Fisher Scientic) were used for F-actin labeling.