Anti ras

Cell lysates were collected in 1X sample buffer (2% SDS, 10% glycerol, 0.01% bromophenol blue, 62.5mM Tris, pH=6.8, 0.1M DTT) and boiled (10 min, 95° C). Protein concentration was determined using Bradford assay (Bio-Rad, cat#5000006). Proteins were resolved using SDS-PAGE gels and transferred to nitrocellulose membranes (GE Healthcare Life Sciences, cat#10600001) as previously described [8 (link)]. Antibodies used include: anti-BRAF (Santa Cruz Biotechnology, cat#sc-5284, 1:1000), anti-RAS (BD Sciences, cat#610001, 1:1000), anti-p16 (Abcam, cat#ab108349, 1:1000), anti-p21 (Abcam cat#ab109199, 1:1000), anti-cyclin A2 (Abcam cat#ab181591, 1:2000), anti-vinculin (Sigma-Aldrich cat#V9131, 1:1000), β-Actin (Sigma-Aldrich, cat#A1978, 1:10000), anti-mouse HRP (Cell Signaling Technology, cat#cst7076, 1:10,000), and anti-rabbit HRP (Cell Signaling Technology, cat#cst7074, 1:5000).

MCF-7 cells were harvested by the use of ice-cold RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) and protein concentration was determined using the BCA protein quantification kit (Beyotime Biotechnology, Shanghai, China); western blotting was performed as previously described [19 (link)]. Primary antibodies of anti-Ac-H3, anti-SOD1, anti-CAT, anti-ERK, anti-p-ERK, anti-Cyt-C, and anti-caspase 9 were purchased from Bioworld Technology (Nanjing, China), anti-PARP, rabbit anti-caspase 3, and anti-Ac-H3 were purchased from Cell Signaling Technology, Danvers, USA, anti-RAS was purchased from BD, USA, and anti-GAPDH was purchased from Earthox, Millbrae, USA. The secondary antibodies were purchased from Biogot Biotechnology (Nanjing, China), and ECL SuperSignal West Femto Maximum Sensitivity Substrate was purchased from Thermo Fisher.

Cell extracts were prepared in IP150 lysis buffer (20 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.5% Nonidet P-40, 10% Glycerol) containing protease inhibitors (1 mM Na₂VO₄, 10 mM NaF, 2 mM PMSF, 5 μg/ml Leupeptin, 10 μg/ml Aprotinin, 1 μg/ml Pepstatin A) (Roche, Switzerland). Equal amounts of protein were separated by SDS-PAGE and transferred onto PVDF membranes (PALL Life Sciences, USA). Membranes were subsequently incubated with the appropriate primary antibodies overnight at 4 °C, followed by incubation with peroxidase-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized using the ECL chemiluminescent detection system (iNtRON Biotechnology, Korea). For immunoprecipitation of protein complexes, cell extracts were precleared with protein G-Sepharose beads (GE Healthcare) and incubated with the appropriate antibodies. Immune complexes were then analyzed by immunoblotting, which was performed using the following antibodies: anti-Ephexin1 and anti-β-actin from Abcam (Cambridge, MA, USA); anti-ERK1/2, antiphospho-ERK1/2 (T202/Y204), anti-Ki67, anti-EGFR, and antiphospho-EphA2 (S897) from Cell Signaling (Danvers, MA, USA); anti-HA, antimyc, anti-V5, and anti-EphA2 from Santa Cruz (Dallas, TX, USA); anti-FLAG (M2) from Sigma-Aldrich; and anti-Ras from BD Biosciences (San Jose, CA, USA).

Cells were lysed in 2× Laemmli buffer at the concentration of 1 × 10⁴ cells/µl. Lysates were treated with 3 μl of Benzonase (25 U/µl, Sigma) for 30 min at 37 °C. RPE-1 and MCF10A cells extracts were a gift of M. Mechali and R. Fernandez de Luco labs respectively (IGH, France). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane and analyzed by western immunoblotting with appropriate antibodies: anti-Claspin (1/50, gift of T. Halazonetis, Geneva), anti-Timeless (1/1000, Interchim), anti-Actin (1/500, Sigma), anti-phospho Ser317-CHK1 or Ser345-CHK1 (1/1000, Cell Signaling Technology, ref 2344S and 2348), anti-CHK1 (1/1000, Cell Signaling Technology, ref 2360), anti-Cdc25A (1/200, Santa Cruz, ref sc-7389), anti-γ-H2AX (1/1000, Millipore, ref 05–636), anti-Tubulin (1/3000, Abcam, ref ab-6161), anti-RPA (1/300, Abcam ref ab-79398), anti-ATR (1/1000, Abcam, ref ab-10312), anti-RAD17 (1/1000, MBL, ref K0120-03), anti H3 (1/3000, Abcam, ref ab-7191), anti-ras (1/500, BD, ref 610002), anti-Actin (1/500, Sigma ref A4700). Blots were incubated with horseradish peroxidase-linked secondary antibody (GE Healthcare) and visualized using the ECL⁺ chemiluminescence method (Pierce).

The cells were lysed with RIPA buffer (#9806, Cell Signaling) supplemented with 1mM PMSF (Sigma-Aldrich). Western blotting was performed using 4–20% gradient SDS-polyacrylamide TRIS-HCl gel (Mini-PROTEAN TGX^®, BioRad) according to the manufacturer’s protocol (Biorad). Antibodies used for western blotting were anti-ras (#610001, 1:1000, BD Bioscience), anti-SV40 large T and small t antigen (#554150, 1:1000, BD Bioscience), anti-β-actin (#A5441, 1:2500, Sigma-Aldrich), and anti-mouse IgG-HRP (#7076S, 1:5000, Cell Signaling).