Anti phospho egfr

Epidermal growth factor (EGF) was purchased from R&D Systems (Minneapolis, MN). We utilized the following antibodies: anti-phospho-EGFR (to phosphotyrosines 1045, 1068, 1148, and 1173), total EGFR, Akt (#4691), phospho-Akt (Thr308, #9275), MAPK (#4695), phospho-MAPK (Thr202/Tyr204, #4370), PI3K (#11889), phospho-PI3K (Tyr458, #4228), PLCγ1 (#5690), phospho-PLCγ1 (Tyr783, #2821), Src (#2123), phospho-Src (Tyr416, #2101), Stat3 (#4904), phospho-Stat3 (Tyr705, #9145), phospho-Stat5 (Tyr694, #4322), Cbl (#2747),and Horseradish peroxidase (HRP)-linked rabbit IgG secondary antibody from Cell Signaling (Danvers, MA);anti-phospho-EGFR (phosphotyrosines845 and 992) from Millipore (Billerica, MA); and Stat5 (sc-835), phospho-Cbl (Tyr700, sc-377571), and goat anti-mouse IgM-HRP (sc-2064) from Santa Cruz Biotechnology (Dallas, TX).

Total cellular lysates were prepared by solubilizing cells using cell lysis buffer containing 1% Triton X-100, 10 mM Tris (pH 7.4), 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, and protease and phosphatase inhibitor cocktail (Sigma-Aldrich). The protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific Inc., Rockford, IL). The lysates were mixed with Laemmli sample buffer for SDS-PAGE.
Nuclear fractions were prepared as described before (31 (link)), cells were washed with ice-cold PBS and scraped into Buffer A (10 mM HEPES (pH 7.5), 10 mM KCl, 2 mM MgCl₂, 1% NP40, and protease inhibitors), passed through a 21-gauge needle, and centrifuged. The pellet (nuclear fraction) was resuspended in Buffer B (20mM HEPES (pH 7.5), 10 mM KCl, 2 mM MgCl₂, 500 mM NaCl and 25% glycerol), homogenized, and kept on ice for 30 min. After centrifugation, the supernatant was mixed with Laemmli sample buffer for SDS-PAGE.
Western blot analysis was performed using anti-EGFR (Millipore, Billerica, MA), anti-phospho-EGFR (Tyr1068), anti-IκBα, anti-phospho-p38, anti-total-p38, anti-phospho-stress-activated protein kinase/c-Jun aminoterminal kinase (SAPK/JNK), anti-total-JNK, anti-total extracellular signal-regulated kinase (ERK)1/2 (Cell Signaling Technology), anti-phospho-ERK1/2 (Promega, Madison, WI), anti-β-actin (Sigma-Aldrich), and anti-Ki67 antibodies.