Phospho p42 44 erk thr202 tyr204

Kwik-Diff stain (9990700) was from Thermo Scientific Shandon. Reagents for priming neutrophils were LPS (E. coli-derived, Sigma, L3024), mouse TNFα (R&D Systems, 410-MT-010), and mouse GM-CSF (Peprotech, 315-03). Reagents for stimulating neutrophils included f-Met-Leu-Phe (fMLP, Sigma, F3506), C5a (Sigma, C5788), and phorbol 12-myristate 13-acetate (PMA, Sigma, P1585). Antibodies for western blotting were Rac1 (clone 23A8, Millipore, 05-389, 1:3000), Rac2 (Millipore, 07-604, 1:5,000), Irak4 (Cell Signaling Technology, 4363, 1:500), MyD88 (Cell Signaling Technology, 4283, 1:500), TLR4 (Cell Signaling Technology, 14358, 1:500), and Trif (Abcam, ab13810, 1:1,000). Antibodies for LPS/TLR4 pathway analysis were from Cell Signaling Technology: phospho-p38 Mapk Thr180/Tyr182 (9211, 1:1000), p38 Mapk (9212, 1:1000), phospho-p42/44 Erk Thr202/Tyr204 (9106, 1:1000), p42/44 Erk (9102, 1:1000), phospho-Akt Thr308 (9275, 1:5000), and Akt (9272, 1:1000). Antibodies for other applications are listed in the relevant sections below. Fc block (553141) was from BD Biosciences.

Ba/F3 cells were grown in RPMI containing 10% fetal bovine serum (FBS) and 15% WEHI3-conditioned medium as a source of IL3. The Ba/F3 cell lines expressing p185^Bcr-Abl with or without Abi1 deficiency were cultured in RPMI containing 10% FBS. The preparation of rabbit polyclonal antibodies against Abi1 and Abi2 has been described previously [41 (link), 42 (link)]. The antibodies against Abl were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and the rabbit monoclonal antibodies for pan- and phospho-Akt (Ser 473), p38 MAPK, phospho-p38 (Thr180/Tyr182), p42/44 ERK, and phospho-p42/44 ERK (Thr202/Tyr204) were obtained from the Cell Signaling Technology, Inc. (Danvers, MA). The monoclonal anti-β-actin antibody and the protease inhibitor cocktail were purchased from Sigma (St. Louis, MO).