Total erk 1 2 antibody

Western blot lysates and preparation were performed as previously described. Immunoprecipitation was achieved using protein G agarose to sepharose beads for preclearing lysates and capturing the immunocomplex after incubation with the immunoprecipitating antibody. In cell line experiments, treatment with inhibitors for the specified number of hours (figure legends) was performed to determine the response of Hsp90, EGFR and its downstream signals to inhibitors. Immunoglobulin G (IgG) and anti-HA tag antibodies were obtained from Abcam. Total EGFR and β-actin antibodies were obtained from Santa Cruz Biotechnology. Total ERK 1/2 antibody was obtained from BD Transduction Laboratories. Phospho-EGFR (pT1068) antibody was purchased from Invitrogen. Hsp90, Hsp70, phospho-AKT (pS473), total AKT, phospho-ERK (pT202/pY204), PARP, and cleaved PARP antibodies were obtained from Cell Signaling Technology. Primary antibodies were diluted 1:1,000, while secondary antibodies were diluted 1:10,000.

suPAR and ATF were purchased from American Diagnostica, Inc. (Greenwich, CT). fMLP, Pertussis toxin (Gαi inhibitor, 100ng/ml), Phosphatidylinositol-specific-phospholipase C (100µg/mL), PD98059 (ERK inhibitor, 25µM) SB203580 (p38^MAPK inhibitor, 10µM), SP600125 (JNK inhibitor, 1µM), EGFR inhibitor ( AG1478, 10µM), ε-aminocaproic acid (EACA; plasmin inhibitor, 100µM), aprotinin (plasmin inhibitor, 100 units/ml) and GM6001 (MMP inhibitor, 10nM) were purchased from Sigma Chemical Co. (St. Louis, MO). Peroxidase-conjugated anti-rabbit IgG antibody (raised in goat) and peroxidase-conjugated anti-mouse IgG antibody (raised in goat) were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Phospho-ERK1/2 antibody was purchased from Promega, Inc. (Madison, WI). Total ERK 1/2 antibody was purchased from BD Transduction Laboratories (Lexington, KY). Phospho-p38^MAPK and phospho-JNK antibodies were purchased from Biosource (Camarillo, CA). Total p38^MAPK and JNK antibodies were purchased from Cell Signaling (Beverley, MA). Dulbecco’s minimal essential medium (DMEM) and Dulbecco’s phosphate buffered saline (dPBS) were purchased from Corning Cellgro (Corning, NY).