Anti phospho egfr tyr1173

Protein samples from total cell lysates (20 or 50 μg) were subjected to electrophoretic separation on a 7.5 or 10% polyacrylamide gel and transblotted onto a polyvinylidine fluoride (PVDF) membrane (Millipore). Blots were blocked at room temperature for 1 hour in Tris-buffered saline (TBS)/Tween 20 (TBS-T) (0.05%) containing 5% bovine serum albumin (BSA) and incubated with: 1:500 rabbit polyclonal anti-EGFR (#2235); 1:500 rabbit monoclonal anti-phospho-EGFR (Tyr1173) (#4407); 1:500 rabbit monoclonal anti-phospho-EGFR (Tyr1068) (#3777); 1:1000 mouse monoclonal antibody anti-phospho-p44/42 MAPK (ERK1/2) (#9106); 1:1000 rabbit polyclonal anti-p44/42 MAPK (ERK1/2) (#9102); 1:1000 rabbit polyclonal anti-phospho-Akt (Thr308) (#9275); 1:1000 rabbit polyclonal anti-Akt (#9272) (Cell Signaling Technology); 1:10000 mouse monoclonal anti-actin (clone C4) (MP Biomedicals). Blots were subsequently washed and incubated with HRP-anti-mouse or HRP-anti-rabbit antibodies at 1:1500 (Dako). Detection was performed using Lumi-Light Western blotting substrate (Roche Diagnostics Nederland B.V.). Images were captured using a digital imaging system (Bio-Rad).

Dulbecco’s modified Eagle’s medium (DMEM)/F-12 and fetal bovine serum were purchased from Thermo Fisher Scientific (Grand Island, NY, USA). BioTrace^TM NT membrane was purchased from Pall Life Sciences (Ann Arbor, MI, USA). The enhanced chemiluminescence (ECL) Western blotting detection system was purchased from Perkin Elmer (Waltham, MA, USA). Anti-phospho-EGFR (Tyr¹¹⁷³, Cat#4407), anti-phospho-p38 MAPK (Thr¹⁸⁰/Tyr¹⁸², Cat#9211), anti-phospho-JNK1/2 (Thr¹⁸³/Tyr¹⁸⁵, Cat#9255), anti-phospho-FoxO1 (Ser²⁵⁶, Cat#9461), and anti-phospho-p65 (Ser⁵³⁶, Cat#3033) antibodies were purchased from Cell Signaling (Danvers, MA, USA). Antibodies of anti-TNFR1 (Cat#sc-52739), anti-TNFR2 (Cat#sc-8041), and p38 MAPK inhibitor (p38i) VIII were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-GAPDH (Cat#MCA-1D4) was purchased from EnCor Biotechnology (Gainesville, FL, USA). AG1478, SP600125, and Tanshinone IIA were purchased from Enzo Life Science (Farmingdale, NY, USA). AS1842856 was obtained from EMD Millipore (Billerica, MA, USA). Helenalin was purchased from Cayman Chemical (Ann Arbor, MI, USA). Recombinant human TNF-α protein was purchased from R&D Systems (Minneapolis, MN, USA). TRIzol reagent, enzymes, and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Opti-MEM I reduced serum medium and G418 were purchased from Invitrogen (Carlsbad, CA, USA) and the X-tremeGENE HP reagent was purchased from Roche (Basel, Switzerland). Cell culture medium and fetal bovine serum were obtained from Hyclone (Beijing, China). Alternative Thioglycollate Medium, Pertussis toxin (PTX), GF109203X (bisindolylmaleimide), Go6983, and tyrphostin A9 were obtained from Sigma (St. Louis, MO, USA), while U0126, tyrphostin AG1478, GM6001, PP2 and Wortmannin were from Calbiochem (La Jolla, CA, USA). Anti-phospho-Akt (Ser473), Anti-phospho-Akt(Thr308), Anti–Akt, Anti–EGFR, Anti–PDGFR, Anti-phospho-EGFR (Tyr1173), Anti-phospho-PDGFR (Tyr1018) and the horseradish peroxidase substrate were bought from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase-conjugated goat anti-rabbit secondary antibody and anti-β-actin antibody were obtained from HuaAn Biotechnology (Hangzhou, China). RIPA lysis buffer and a BCA kit were bought from Beyotime (Haimen, China).