Mouse anti wasp d1

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 were centrifuged, supernatant was cleared, and the cells were re-suspended in lysis buffer as previously described [9 (link),33 (link),34 (link)]. Cells were placed on ice for 30 min and then centrifuged. The cell lysate was collected and moved to fresh tubes. Sample buffer was then added to the cell lysates and samples were heated for 5 min at 100 °C and then centrifuged at 10,000× g.
Western blots were performed as described [6 (link),9 (link),34 (link)].
The primary antibodies for WB, PTP, and Imaging were: Rabbit anti-pVAV1 (Y160) (Bio Source Cat# Bs44482, Invitrogen, Thermofisher); human anti-CD28 (BioLegend, London, United Kingdom Cat# 302934); Rabbit anti-SHP-1 (C-19) (Santa-Cruz Biotechnology, Heidelberg, Germany, Cat# sc-287); Mouse anti-GAPDH (0411) (Santa-Cruz Biotechnology, Heidelberg, Germany, Cat# sc-47724); and Mouse anti-WASp (D1) (Santa-Cruz Biotechnology, Heidelberg, Germany, Cat# sc-5300).
Secondary Antibodies were: Goat anti-Rabbit (Santa Cruz Biotechnology, Heidelberg, Germany, Cat# sc-2004) and Goat anti-Mouse (Jackson ImmunoResearch, West Grove, PA, USA, cat# 115-035-003).