Mouse anti cd71

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.

Proteins were separated by SDS-PAGE and electrotransferred onto nitrocellulose, incubated with antibodies, and processed for detection by chemiluminescence as described previously (19 , 29 (link), 40 ). The following antibodies were used: sheep anti-DGKε (R&D Systems, catalog no.: AF7069), mouse anti-Myc (ThermoFisher, catalog no.: R950-25), mouse anti-CD71 (transferrin receptor, Santa Cruz Biotechnology, catalog no.: sc-32272), rabbit anti-Jak1 (Cell Signaling, catalog no.: 3344), mouse anti-actin (BD Transduction Laboratories, catalog no.: 612657), rabbit anti-flotillin-2 (Cell Signaling, catalog no.: 3436), donkey anti-sheep IgG-HRP (Jackson ImmunoResearch, catalog no.: 713-035-003), mouse anti-HA IgG-HRP (Cell Signaling, catalog no.: 2999), goat anti-mouse IgG-HRP (Jackson ImmunoResearch, catalog no.: 115-035-146), and goat anti-rabbit IgG-HRP (Merck, catalog no.: 401315 or Rockland, catalog no.: 611-1302). All the antibodies used are listed in supplemental Table S2. Visualized bands were analyzed densitometrically using the ImageJ program. Absorbance values were corrected by background subtraction, which in the case of click chemistry included samples incubated with DMSO.