Rabbit anti giantin

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.

The following primary antibodies were used for Western blotting and immunofluorescence and were kindly provided: mouse anti-gD (LP14) and mouse anti-gB (R69), from Tony Minson (University of Cambridge); mouse anti-VP16 (LP1), from Colin Crump (University of Cambridge); mouse anti-gE, from David Johnson (Oregon Health and Science University); and mouse anti-nectin1 (CK6), from Claude Krummenacher (Rowan University, NJ). Our rabbit VP22-specific antibody (AGV031) was described previously (68 (link)). Commercially available antibodies used in this study included mouse anti-V5, mouse anti-gC, mouse anti-β-actin, mouse anti-CD63, rabbit anti-giantin, mouse anti-CHMP4A, and rabbit anti-CHMP4B (all from Abcam); mouse anti-CD71 (Santa Cruz); and mouse anti-γ-tubulin and mouse anti-α-tubulin (Sigma). Goat anti-mouse IRDye 680RD and goat anti-rabbit IRDye 800CW (Li-Cor Biosciences) secondary antibodies were used as appropriate for Western blotting, while Alexa Fluor-conjugated secondary antibodies (Invitrogen) were used for immunofluorescence. Endocytic structures were labeled by incubating cells with Texas Red-conjugated transferrin (Invitrogen) or Alexa Fluor 488-conjugated transferrin (Molecular Probes) at a concentration of 1 μg/ml for 30 min. Brefeldin A (Sigma) was used at a concentration of 1 μg/ml.