Anti p egfr

Western blots were performed as previously described21 (link), and the following antibodies were used: anti-EGFR (1:1000, Ca# 4267), anti-p-EGFR (1:1000, Ca# 3777), anti-JAK2 (1:1000, Ca# 3230), anti-p-JAK2 (1:1000, Ca# 3776), anti-STAT3(1:1000, Ca# 9139), anti-p-STAT3(1:1000, Ca# 4093), anti-α-actinin(1:1000, Ca# 6487) (Cell Signalling Technology, USA). Antibody against NPNT was obtained from Abcam (1:1000, Ca# ab64419). anti-EGFR neutralizing, clone LA1 (2 μg/mL, Ca# 05-101) was purchased from Sigma and EGFR dominant negative plasmid (pRK5RS-HERCD533) was obtained from Addgene.

Immunoblots were used to analyze the protein expression levels of STAT3 as previously described [6 (link), 7 (link)]. UM-SCC-1, −5 and transfected STAT3-2.4 and NEG4.17 cells were assessed for STAT-3, phosphorylated STAT-3 and GAPDH. Cell lysates were prepared and equal amounts of protein were loaded in each gel lane. Separation was performed by 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to Immobilon-P membrane (Millipore Corp, Bedford, MA). The immunoblots were blocked in 10 % milk–Tris-buffered saline with Tween-20 (TBS-T) (20 mmol/L Tris HCL [pH 7.5], NaCl 137 mmol/L, and 0.05 % Tween-20) for 1 h at room temperature. The primary antibodies of anti-STAT-3, anti-p-STAT-3 (Cell Signaling Technologies, Beverly, MA), anti-EGFr (Sigma-Aldrich, St. Louis, MO), anti-p-EGFr (cell signaling technologies, Beverly, MA) and anti-GAPDH (Santa Cruz Technologies, Inc., Santa Cruz, CA) were incubated overnight at 4 ° C with 2 % milk–TBS-T. The secondary antibody, anti-mouse–IgG–horseradish peroxidase antibody (Sigma Chemical Company, St. Louis, MO) was incubated at room temperature for 1 h. The blots were developed by chemiluminescence (Amersham Life Sciences, Inc., Arlington Heights, IL).

Whole cell lysates were extracted using lysis buffer (50 mM Tris-HCL pH 8.0, 50 mM Tris-HCL pH 8.0, 150 mM NaCl, 5 mM MgCl2, 1% TritonX-100, 0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate, 40 mM sodium fluoride, and 1 mM sodium orthovanadate) containing phosphatase inhibitors, protease inhibitors and 20 mM DTT, as reported previously [14 (link)]. Western blot analysis was carried out by conventional methods using the following primary antibodies: anti-HER2, anti-phospho(P)-HER2 (Tyr1221/1222), anti-EGFR, anti-P-EGFR (Tyr1068), anti-HER3, anti-P-HER3 (Tyr1289), anti-Akt, anti-P-Akt (Ser473), anti-p44/p42 MAPK, anti-P-p44/p42 MAPK (Thr202/Tyr204), anti-β-actin (used as a loading control) (Cell Signaling Technology, Danvers, MA, USA) and anti-FLAG® M2 (Sigma-Aldrich, St. Louis, MO, USA). As secondary antibodies HRP-conjugated anti-mouse or anti-rabbit IgG (Cell Signaling Technology, Danvers, MA, USA) were used. Proteins were detected using a C-Digit® imaging system (LI-COR Biosciences, Lincoln, NE, USA) and were visualized using Image Studio™ for C-Digit®. A dimerization assay was performed as reported previously [11 (link)]. The cell lysate preparation and subsequent procedures were the same as indicated above, expect that DTT (reducing agent) was not included in the lysis buffer.

Cells were harvested and lysed in SDS lysis buffer (PMSF, Protease, and Phosphatase Inhibitor Cocktail added) for 30 min at 4°C. Total protein was extracted from tissues with T-PER Tissue Protein Extraction Reagent (Pierce, Rockford, IL, USA) according to the manufacturer's protocol. The proteins were dissociated and separated by SDS/PAGE and then transferred to PVDF membranes, which were incubated with primary antibodies. The primary antibodies used for western blotting and their sources were as follows: anti-SEMA3C (Proteintech, 19242-1-AP), anti-GAPDH (Proteintech, 60004-1-Ig), anti-p-ERK1/2 (Cell Signal Technology, #4370), anti-ERK1/2 (Cell Signal Technology, #4695), anti-p-EGFR (Cell Signal Technology, #3777), anti-EGFR (Proteintech, 18986-1-AP), anti-p-Her2 (Cell Signal Technology, #2244), anti-Her2 (Cell Signal Technology, #2244), anti-Her2 (Proteintech, 18299-1-AP), anti-p-MET (Cell Signal Technology, #3077), anti-MET (Proteintech, 25869-1-AP), anti-p-SRC (Cell Signal Technology, #6943), and anti-SRC (Proteintech, 11097-1-AP). Antigen-antibody complexes were detected using horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology #7074; #7076) with enhanced chemiluminescence (ECL) western blot detection reagent (Merck Millipore) according to the manufacturer's protocol.

Protein lysates (20–30 μg) were loaded onto 10–12% SDS-PAGE gels, electrophoresis, and blocking were conducted as described16 (link). Blots were probed overnight at 4 °C with respective antibodies. Primary antibodies (anti-p-Akt, anti-p-EGFR, anti-Bcl-XL, anti-Ku80, anti-p-STAT3, STAT3, anti-p-H2AX, and anti-β-actin) were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-EGFR and anti-Rad51 antibody was purchased from Santa Cruz Biotechnology, Dallas, TX, USA, and anti-EphB4 antibody (clone m265) was provided by Vasgene Therapeutics Inc. (Los Angeles, CA, USA). Horseradish peroxidase (HRP)–conjugated secondary antibodies were obtained from Sigma (St. Louis, MO, USA).

Proteins were separated by 10% SDS-containing polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride microporous membrane (Millipore, MA, USA). The membrane was probed with primary antibodies including HAb18, C-19 (Santa Cruz Biotechnology), anti-MMP-2 (Santa Cruz Biotechnology), anti-p-ERK1/2, anti-p-FAK, anti-p-Akt, anti-p-EGFR, anti-ERK1/2, anti-Akt, anti-EGFR (Cell Signaling Technology, Danvers, USA), anti-FAK (BD), and anti-α-tubulin antibodies (Santa Cruz Biotechnology).