Alexa fluor 488 conjugated goat anti mouse igg1 antibody

Antibodies were obtained from the indicated suppliers. The following primary antibodies were used for western blotting: mouse anti-GFP 1:500 (Roche Applied Science—Catalogue no. 11 814 460 001), mouse anti-WASp D1 1:250 (sc-5300) and rabbit anti-GAPDH 1:1000 (Santa Cruz—sc-47724). HRP-conjugated goat anti-mouse 1:10,000 (Jackson—115-035-003) was used as a secondary antibody. Proteasome activity was blocked by addition of MG132 (Sigma–Aldrich—C2211) at a final concentration of 50 µM for 3 h before the cells were harvested. The following primary antibodies were used for flow cytometry: mouse anti-WASp D1 1:250 (Santa Cruz—sc-5300). The KIM127 hybridoma was purchased from the ATCC (KIM127 (ATCC® CRL2838™)) and the supernatant was purified to obtain the antiactivated human LFA-1 antibody (Sigma–Aldrich). For flow cytometry, secondary Alexa Fluor 488-conjugated goat anti-mouse IgG1 antibody was used 1:2500 (Jackson – A11034). SMC #13 was synthesized and purified (>90% purity) using LC-MS/MS by ChemBridge Research Laboratories LLC.

Cells (1 × 10⁶/ml) were either stimulated with 2.5 ng/ml PMA and 1 μg/ml ionomycin or left unstimulated. Cells were collected (5 × 10⁵ cells per sample), washed twice with PBS, and fixed with 0.4 ml 3.7% paraformaldehyde for 20 min at room temperature. After two additional washes, cells were permeabilized with 0.1% Triton X-100 in PBS for 4 min at room temperature. Cells were blocked for 45 min with 2% goat serum, followed by 45 min incubation with 0.1 μg mouse anti-WASp Ab, on ice. The samples were washed twice, and then incubated with 1:2000-diluted Alexa Fluor 488-conjugated goat anti-mouse IgG1 antibody (Jackson), for 45 min on ice. Fluorescence was analyzed by flow cytometry on a FACS Gallios system (Beckman Coulter).