Donkey anti rabbit igg

The mouse anti-CI-MPR antibody (Ab2733; Abcam) and rabbit anti-CI-MPR antibody (ab124767; Abcam) were used for immunofluorescence and Western blot, respectively. The antibodies for Giantin (924,302; Convance), HRS (ab155539; Abcam), GM130 (610,822; BD Biosciences) and EEA1 (610,456; BD Biosciences) were used for immunofluorescence. The antibodies for γ1 (610,386; BD Biosciences), γ2 (HPA004106; Sigma-Aldrich), μ1A (AB111135; Abcam), GAPDH (G9545; Sigma-Aldrich) and β-actin (sc-47778; Santa Cruz) were used for Western blot. The same antibodies for γ1 and γ2 were also used for immunofluorescence. The rabbit anti-Vps26 and anti-Snx2 anti-serum were kind gifts from Juan Bonifacino, and previously described (97 (link), 98 (link)). The rabbit anti-GFP antibody used for western-blot was a gift from R. Hegde (MRC). The mouse monoclonal anti-CD8 antibody used for flow cytometry and antibody uptake assays was a kind gift from Matthew Seaman (MRC) (9 (link)). Horseradish-peroxidase-conjugated donkey anti-mouse immunoglobulin G (IgG) and donkey anti-rabbit IgG were obtained from GE Healthcare. Secondary antibodies conjugated to Alexa fluorophores were purchased from Thermo Scientific.

HEK293 cells were seeded in six-well plates and transfected with either HA-CXCR4-WT or HA-CXCR4-W195L. 48 h later, cells were washed with phosphate-buffered saline (PBS) and serum starved in HBSS for 2 h. Cells were then stimulated with 200 nM CXCL12 or vehicle for 30 min before washing with ice-cold PBS and lysed with RIPA lysis buffer (50 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS complemented with Halt™ Protease Inhibitor Cocktail (Thermo Fischer) and PhosSTOP phosphatase inhibitor (Roche)). Cell lysates were cleared by centrifugation at 14 000 rpm for 15 min at 4 °C and the supernatant was mixed with 2X SDS sample buffer. Samples were then analyzed by electrophoresis on a 10% SDS-polyacrylamide gel, transferred to a PVDF membrane, and immunoblotted with the following antibodies: pS324/pS325-CXCR4 phospho-CXC Chemokine Receptor 4 rabbit 1:1000 (7TM antibodies), anti-HA 3F10 rat monoclonal 1:1000 (Roche) and anti-GAPDH rabbit monoclonal 1:5000 (Cell Signaling) antibodies. Membranes were then washed and incubated with HRP-coupled secondary donkey anti-rabbit IgG (GE healthcare) or goat anti-rat IgG (Sigma) antibodies, and the images were acquired and analyzed using a Chemidoc Imaging System (Bio-Rad). The unprocessed scans of the Western blots are presented in Supplementary Fig. 16.

Whole-cell lysates were prepared by incubating cells for 30 min in cold radioimmune precipitation assay buffer with Complete Protease Inhibitor Cocktail Tablets (Roche Diagnostics, Tokyo, Japan). Samples were then centrifuged at 10,000× g to allow the cell debris to settle, and 30 µg total protein from the resulting supernatant was separated by 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electro-transferred onto membranes, probed with 1:500 (anti-Kv2.1 Ab) or 1:1000 (anti- β-actin Ab) of the indicated primary antibodies, and then probed with 1:8,000 of donkey anti-rabbit IgG (GE Healthcare, Piscataway, NJ) or sheep anti-mouse IgG (GE Healthcare) conjugated with horseradish peroxidase. Blots were visualized with enhanced Chemi-Lumi One Super (Nacalai Tesque, Kyoto, Japan) (Daijo et al., 2016 (link); Tanaka et al., 2011b (link)). Experiments were performed in triplicate, and representative blots are shown. Detailed protocols are available at protocols.io (10.17504/protocols.io.x9mfr46).