Phospho erk1 2 d13.14.4e

BMM were either non-treated or treated with CpG (1 μg/ml) in the presence or absence of mCD47.ex for 30min at 37°C. Thereafter, BMM were washed and then incubated with ice-cold lysis buffer containing 25mM Tris-HCl, pH 7.4, 150mM NaCl, 1% Triton X-100, protease inhibitors (Protease Inhibitor Cocktail, Sigma-Aldrich), phosphatase inhibitors (Phosphatase Inhibitor Cocktail 1 and 2, Sigma), 3mM PMSF and 2mM pervanadate. After centrifugation at 12,000rpm for 5min, cell lysates were collected and then heated at 95°C for 3min in SDS-PAGE sample buffer. After electrophoresis in acrylamide gels, proteins were transferred onto nitrocellulose, followed by blocking with 3% BSA and probed for: SIRPα (clone P84, BioLegend); phospho-Erk1/2 (D13.14.4E); phospho-p38 (D3F9); and beta-actin (13E5) (all from Cell Signaling Technology). Densitometric analyses were performed using Image J (NIH).

Cells were washed in cold PBS and lysed directly in Laemmli sample buffer. Lysates were resolved by SDS polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 1% BSA and incubated with indicated primary antibodies overnight at 4°C. Western assays were detected using the horseradish peroxidase-conjugated secondary antibodies followed by development using chemiluminescence substrate (Pierce, Rockford, IL). Primary antibodies used were: ERK2 (D-2) from Santa Cruz Biotech. Inc. (Santa Cruz, CA); ERBB3 (1B2E), phospho-ERBB3 Y1197 (C56E4), phospho-ERBB3 Y1289 (21D3), ERBB2 (D8F12), phospho-ERBB2 Y1196 (D66B7), MET, phospho-MET Y1234/1235 (D26), PDGFR, phospho-PDGFRβ Y751 (C63G6), EGFR, phospho-EGFR Y845, IGF1R, phospho-IGF1Rβ Y1131, AKT, phospho-AKT T308 (C31E5E), phospho-AKT S473 (D9E) and phospho-ERK1/2 (D13.14.4E), and phospho-TSC2 T1462 from Cell Signaling Tech. (Danvers, MA); Actin from Sigma-Aldrich (St Louis, MO). Chemiluminescence was visualized on a Versadoc MultiImager and quantitated using Quantity-One software (Bio-Rad, Hercules, CA).

Cells were washed in cold PBS and lysed directly in Laemmli sample buffer. Lysates were resolved by SDS polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 1% BSA and incubated with indicated primary antibodies overnight at 4°C. Proteins were detected using the horseradish peroxidase-conjugated secondary antibodies followed by development using chemiluminescence substrate (Pierce, Rockford, IL). The following primary antibodies were used: ERK2 (D-2), Cyclin A and Noxa from Santa Cruz Biotech. Inc. (Santa Cruz, CA); Bcl-2, Bcl-xl, Mcl-1, Bax, Bad, Bid, Puma, cleaved caspase 3, cleaved PARP, RB, phospho-RB (S780), cMET, phospho-cMET Y1234/1235 (D26), phospho-cMET Y1349, AKT, phospho-AKT T308 (C31E5E), phospho-AKT S473 (D9E), phospho-ERK1/2 (D13.14.4E), Stat3, phospho-Stat3 Y705, p110α-PI3K, p110β-PI3K and p110δ-PI3K from Cell Signaling Tech; cyclin D1 and Bcl-w from BD Pharmingen, p110δ-PI3K, α-SMA and FAP from Abcam (Cambridge, MA); Bim-EL and BMF from Enzo Life Sciences (Farmingdale, NY); Actin from Sigma-Aldrich (St Louis, MO) and Hsp90 (clone 4F3.E8) from StressMarq Biosciences (Victoria BC). Chemiluminescence was visualized on a ChemiDoc Imaging System and quantitated using Image Lab 4.1 software (Bio-Rad, Hercules, CA).