Anti egfr 2232

Mouse monoclonal anti‐SHP2 (B‐1, sc‐7384), anti‐PDGFRβ‐pY716 (F‐10, sc‐365464), and anti‐ERK2 (D‐2, sc‐1647) antibodies, as well as rabbit polyclonal anti‐PDGFRβ (958, sc‐432) and anti‐PDGFRβpY579 (sc‐135671) antibodies, were purchased from Santa Cruz Biotechnology. Mouse monoclonal anti‐MEK1 (61B12, #2352), rabbit monoclonal anti‐PDGFRβpY857 (C43E9, #3170) and anti‐PDGFRβpY1009 (42F9, #3124) antibodies, as well as rabbit polyclonal anti‐pMEK S217/221 (#9121), anti‐pERK1/2 T202/Y204 (#9101), and anti‐EGFR (#2232) antibodies, were purchased from Cell Signaling. Mouse monoclonal anti‐phosphotyrosine antibody cocktail (4G10 platinum, 05‐1050) was purchased from Millipore. All antibodies were used at the concentrations recommended by their manufacturers.

Total proteins were extracted in Ripa buffer, and 30 ug of proteins was loaded for Western blot analyses, performed on SDS-PAGE gels under denaturing conditions. For the analysis of Beclin1, LC3 and p62 proteins, 12% bis-acrylamide gels were employed, whereas EGFR proteins were separated on Nu-PAGE 4–12% Bis-Tris Gel (Invitrogen™, Thermo Fisher Scientific, Waltham, MA, USA). Proteins were then electrotransferred onto a nitrocellulose membrane, and nonspecific protein binding was blocked using 10% Blotto-0.1% Tween PBS or 5% BSA-0.1% Tween PBS, respectively. Membranes were incubated with primary antibodies (anti-LC3, L8918, Sigma Aldrich; anti-p62, 610832, BD Biosciences; anti-Beclin1, ab207612, Abcam; anti-EGFR, #2232, Cell Signaling; anti-β-actin A2066 Sigma-Aldrich) diluted 1:1000 in the blocking solution, overnight at 4 °C. Horseradish peroxidase-conjugated secondary antibodies (antimouse LNA931V, GE Healthcare, antirabbit LNA934V, GE Healthcare) were used to detect the primary antibodies, and blot density was quantified with ImageJ™ software (NIH, Bethesda, MD, USA). Protein band quantifications were normalized on the total protein band at 48 KDa (Ponceau Red band) or on β-actin, according to the most appropriate technique for the experiment.

The following antibodies were purchased: anti-BRD4 (E2A7X) from Cell Signaling Technology, anti-BRD2 (D89B4) from Cell Signaling Technology, anti-BRD3 (2088C30) from Abcam Technology, anti-c-Myc (A19032) from ABclonal Technology, anti-c-Met (25869-1-AP) from Proteintech Technology, anti-EGFR (2232) from Cell Signaling Technology, and anti-GAPDH (AP0063) from Bioworld Technology Inc. All antibodies were utilized at 1:1000 dilutions, except GAPDH (1:10,000). JQ1 (HY-13030) and (2-Hydroxypropyl)-β-cyclodextrin (HY-101103) were purchased from Haoyuan Chemexpress Co., Ltd. Trichostatin A (T6270) was purchased from Target Mol, Inc. Vorinostat (T1583) was purchased from Target Mol, Inc. Osimertinib (HY-15772) was purchased from MedChemExpress (Monmouth Junction, NJ, USA). Reagents were dissolved in DMSO at 20 mmol/L, stored at -20℃, and diluted just before use. Lipofectamine 2000® transfection reagent (11668-019) was purchased from Life Technologies Co. Invitrogen (Carlsbad, CA, USA).