Rap1b

To examine protein expression in various cellular domains, neutrophils were either maintained in suspension without stimulation or stimulated with fMLP in suspension or plated on fibrinogen-coated/anti-CD11b–coated plates for specific times, as indicated in the figures. WCL was performed using standard triton-based lysis buffer. Triton insoluble or DRMs were prepared as described before (Kumar et al., 2012 (link)). Cell lysates containing equal amounts of protein were separated by SDS-PAGE and probed for p-Akt, Akt, p-p38, p38, p-Erk, ERrk, and Rap1b (all from Cell Signaling Technology, Boston, MA), actin (Sigma-Aldrich), CD11b (Abcam), and Rap1 (BD). For immunoprecipitation, rested or stimulated WT and Rap1b^−/− neutrophils were lysed in 1% NP-40 lysis buffer and 500 µg protein was subjected to each immunoprecipitation with 2 µg anti–SHP-1 antibody, followed by protein A/G agarose beads. Blots were probed with anti–phospho-tyrosine 4G10 antibody, followed by anti–SHP-1 (Santa Cruz Biotechnology, Inc.) antibody.

Antibodies against αL/LFA-1 (2D7), αM/Mac1/CD11b (M1/70), β2 (C17/16), and Rap1 were obtained from BD. Human anti–ICAM-1 (HA58) and β1 (eBioHmb1-1) were obtained from eBioscience. Phospho -Akt (Ser473), Akt, p-p38, p38, p-Erk, Erk, Rap1b, and anti–VE-cadherin (clone 55-7H1) were purchased from Cell Signaling Technology. Anti–SHP-1 antibody was obtained from Santa Cruz Biotechnology, Inc. Rhodamine-labeled phalloidin, IgG conjugated to Alexa Fluor 488, Alexa Fluor 594, Alexa 6Fluor 47, or Cy5 were obtained from Invitrogen. Anti-PIP3 antibody and PTEN inhibitor SF-1670 were from Echelon Biosciences. Antibody used for immunoblotting of CD11b was from Abcam, Src-inhibitor PP2, SHP-1/2 inhibitor NSC87877, and serine-protease inhibitor DFP were obtained from EMD Millipore. Akt-inhibitor MK2206 was obtained from Selleckchem. Rac inhibitor NSC23766 was a gift from Y. Zheng (Cincinnati Children’s Hospital Medical Center, Cincinnati, USA). Anti–β-actin antibody and PI3K inhibitor Ly294002 were obtained from Sigma-Aldrich.