Phospho faktyr925

Experiments were performed at a cellular density of 60% to 70%. Unless otherwise indicated, cells were incubated with increasing doses of INC280 (100, 500, 1000 nM) for 4 or 24 hours before stimulation with HGF (50 ng/ml) for 15 minutes. Whole-cell lysates were prepared as described before [17 (link)]. Membranes were sequentially probed with antibodies against phospho-Akt^Ser473, Akt, phospho-ERK^{Thr202/Tyr204}, ERK, phospho-cMET^Tyr1349, cMET, phospho-FAK^Tyr925, FAK (Cell Signaling, Beverly, MA), HIF-1α (Novus Biologicals, Littleton, CO) and β-actin (Santa Cruz Biotechnologies, Santa Cruz, CA). For detection of HIF-1α, MiaPaCa2(G250) cells were incubated for 24 hours with INC280 (500 nM) ± DFX (100 μM) as described [19 (link)]. Antibodies were detected by enhanced chemiluminescence (Amersham Bioscience, Piscataway, NJ).

3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) (5 mg/mL; Sigma, St. Louis, MO, USA), dimethyl sulfoxide (DMSO; Sigma). Afatinib (BIBW2992) was purchased from Selleck Chemicals (Houston, TX). All these compounds were dissolved in DMSO at 10 mmol/L as stock solutions and stored at -20°C. Antibodies against GAPDH were from Epitomics (Burlingame, CA). Antibodies against CCR7 (Y59), VEGFR3, VEGFR2, VEGFC, and LYVE-1 were from Abcam (Cambridge, MA, USA). Antibodies against phospho-EGFR (Y1068), EGFR, Akt, phospho-Akt (S473), JAK1/2, phospho-JAK1 (Tyr1022/1023), phospho-JAK2 (Tyr1007/1008), c-Myc, STAT3, phospho-STAT3 (Tyr705), FAK, and phospho-FAK (Tyr925) were purchased from Cell Signaling Technology (Beverly, MA).

Western blot was performed using the following primary antibodies: phospho-Akt (Ser 473) and phospho-Fak (Tyr 925) (Cell Signaling Technology, Danvers, MA, USA). Briefly, splenocytes from the RABV-infected and mock-infected mice were harvested, and splenocytes from the mock-infected mice were stimulated with or without 60 ng/ml CCL5 at 37°C for 5 min. After being washed with PBS, the cells were lysed using SDS-loading buffer and boiled for 10 min. Lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred thereafter to a nitrocellulose membrane. After being blocked in TBST with 5% BSA for 1 hour (h), the membrane was incubated with primary antibodies overnight at 4°C, followed by incubation with horseradish peroxidase-labeled secondary antibody. Detection was performed with ECL substrate (Thermo Scientific, West Palm Beach, FL, USA).