Anti phospho erk1 2 t202 y204

Cell media and chemicals, unless otherwise stated, were obtained from Sigma (St. Louis, MO). Fetal bovin serum (FBS), horse serum (HS), and NGF were obtained from Invitrogen Laboratories (Paisley, U.K.). NGF was used at a concentration of 50 ng/ml (approximately 4 × 10⁻⁹ M). Anti-phospho-TrkA (Y490) (Cat # 9141), anti-TrkA (Cat # 2505), anti-phospho-ERK1/2 (T202/Y204) (Cat # 9106), anti-ERK (Cat # 9107), anti-phospho-AKT (S473) (Cat # 4051), anti-AKT (Cat # 4685), anti-phospho-CREB (S133) (Cat # 9191), anti-CREB (Cat # 9197), and anti-P75^NTR (Cat # 4201) antibodies were from Cell Signaling Technology (Danvers, MA). Anti-Grb2 antibody (Cat # sc-17813) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rat pheochromocytoma (PC12) cells were obtained from the American Type Culture Collection (Manassas, VA), and cultured, at passages between the 5 and 20, in RPMI-1640 (GIBCO), supplemented with 10% horse serum (HS), 5% fetal bovine serum (FBS), 2 mM L-glutamine, 50 IU/ml penicillin, and 50 μg/ml streptomycin.

Cells were lysed with RIPA lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate) supplemented by protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktails (Calbiochem). Lysates were separated on 7.5% or 8% Tris-Glycine SDS-polyacrylamide gel and were transferred to PVDF membranes (Millipore). The membranes were blocked with 5% skim milk (Bio-Rad) dissolved in TBST buffer (50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20). Then, the membranes were incubated with primary antibodies overnight at 4°C. Anti-EGFR antibody (#A300-388A) was purchased from Bethyl Laboratories. Anti-β-actin antibody (#A5441) and anti-γ-tubulin antibody (#A9044) were purchased from Sigma-Aldrich. All other antibodies including anti-phospho EGFR Y1068 (#3777), anti-phospho ERBB2 Y1221/1222 (#2243), anti-ERBB2 (#2165), anti-phospho SRC Y416 (#6943), anti-SRC (#2109), anti-phospho-ERK 1/2 T202/Y204 (#4370), anti-ERK 1/2 (#4695), anti-phospho AKT S473 (#4060), and anti-AKT (#9272) were purchased from Cell Signaling Technologies. Horseradish peroxidase-conjugated secondary antibodies (anti-rabbit: #31460, anti-mouse: #31430, Pierce) and SuperSignal West Pico Chemiluminescent Substrate (Pierce) were used to detect signals.

Retinas were homogenized in lysis buffer (20 mM Tris [pH 8.0], 135 mM NaCl, 1% sodium dodecyl sulfate [SDS], and 10% glycerol supplemented with protease inhibitors) and centrifuged at 14,000 rpm for 5 min. The supernatants were collected and diluted in Laemmli sample buffer 5× (4% SDS, 10% glycerol, 0.004% bromophenol blue, 0.1 M dithiothreitol, and 0.125 M Tris, pH 6.8). Extracts were processed using standard sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting procedures using precast Mini-PROTEAN TGX gels (Bio-Rad). Equal volumes were loaded on the gels. Antibodies include anti-acetyl p53 K382 (2570, Cell Signaling), anti-acetyl p53 K381 (ab61241, abcam), anti-c-Jun (9165, Cell Signaling), anti-Erk1/2 (9102, Cell Signaling), anti-Fas (ab82419, abcam), anti-JNK (9252, Cell Signaling), anti-p21 (sc-397, Santa Cruz), anti-p53 (MAB1746, R&D systems), anti-Phospho-Erk1/2 T202/Y204 (9255, Cell Signaling), anti-Phospho-JNK T183/Y185 (9255, Cell Signaling), anti-Phospho-c-Jun s63 (2301, Cell Signaling), anti-Phospho-p53 Ser15 (9286, Cell Signaling), anti-PUMA (7467, Cell Signaling), and anti-α-tubulin (T5168, Sigma). Secondary antibodies were obtained from Southern Biotech and Jackson ImmunoResearch. Quantity One software (Bio-Rad) was used for quantification.

The following reagents and materials were used. From Cell Signaling technology (Danvers, MA, USA): anti-APP (ref. 2450), anti-FAK (ref. 3285), anti-phospho-FAK (Y576/577) (ref. 3281), anti-Akt (ref. 4685), anti-phospho-Akt (S473) (ref. 4060), anti-PDK1 (ref. 3062), anti-phospho-PDK1 (S241) (ref. 3061), anti-phospho-PKC pan (T514) (ref. 9379), anti-p38 MAPK (ref. 9212), anti-phospho-p38 MAPK (T180/Y182) (ref. 9211), anti-MEK1/2 (ref. 9126), anti-phospho-MEK1/2 (S217/221) (ref. 9154), anti-ERK1/2 (ref. 9102), anti-phospho-ERK1/2 (T202/y204) (ref. 4370), anti-CREB (ref. 9104), anti-IκBα (ref. 4814S), anti-phospho-IκBα (S32) (ref. 2859), anti-NFκB p65 (ref. 8242), anti-phospho-NFκB p65 (ref. 3033S), anti-SEK1 (ref. 9152), and anti-phospho-SEK1 (S257/T261) (Ref. 9156). Anti-PKC-pan was from SigmaAldrich (St Louis, MO, USA) (ref. SAB4502356). Electrophoresis reagents were purchased from Bio-Rad (Hercules, CA, USA) and trypsin from Promega (Madison, WI, USA).

CRM646-A was prepared as previously described [1 (link)]. Paclitaxel and PD98059 were purchased from Calbiochem. A23187 and gramicidin were purchased from Sigma-Aldrich and Thermo Fisher Scientific (USA), respectively. Hoechst 33342, propidium iodide, FM1-43FX, fluorescent Mg²⁺ indicator (mag-fura-2), fluorescent Na⁺ indicator (CoroNa Green), and fluorescent Ca²⁺ indicator (Fluo-4) were purchased from Thermo Fisher Scientific. Anti-ERK1/2 antibody and anti-phospho-ERK1/2 (T202/Y204) were purchased from Cell Signaling Technology. Methanol (MeOH), dimethyl sulfoxide (DMSO), 3.7% formaldehyde, Triton X-100, bovine serum albumin (BSA), trypan blue solution, and Tween-20 were purchased from Sigma-Aldrich.