Anti erk

All cells were lysed in 1% NP40 buffer (150 mM NaCl, 50 mM Tris-HCl pH 8, 10% glycerol, 2 mM EDTA) supplemented with 1 mM cOmplete Mini protease inhibitors (Roche) and 1 mM sodium orthovanadate (ThermoFisher). Proteins were separated on a 4–20% SDS-PAGE gel (BioRad) and transferred to polyvinylidene difluoride (PVDF) membrane. Membrane was incubated in 5% BSA and 0.1% Tween-20 in PBS for 1 hr, incubated overnight at 4°C with primary antibodies (1:10000 anti-β-actin, Santa Cruz #sc-47778; 1:1000 anti-SHC, BD Transduction #610878; 1:1000 anti-ERK, Invitrogen #13–6200; 1:1000 anti-phospho-SHC (Tyr239/240), Cell Signaling #2434; 1:2000 anti-phospho-p44/42 ERK (Thr202/Tyr204), Cell Signaling #4370; 1:1000 anti-EGFR, Cell Signaling #2232; 1:700 anti-phospho-Thr/Tyr, Cell Signaling #9381; 1:500 anti-CLDN1, Invitrogen #37–4900; 1:350 anti-OCLN, Invitrogen #33–1500), then incubated for 1 hr at room temperature with horseradish peroxidase-conjugated secondary antibodies (1:10000 anti-rabbit, Cell Signaling #7074; 1:10000 anti-mouse, Cell Signaling #7076). Membrane was added SuperSignal West Pico PLUS Chemiluminescent or West Femto Maximum Sensitivity substrate (ThermoFisher) and exposed to CL-XPosure film (ThermoFisher).

Cells were plated in 6 wells at a density of 2 × 10⁵ cells per well. After PARP inhibition, cells were washed twice with PBS and resuspended in 200 μl of TR3 Lysis Buffer (3% SDS, 10% Glycerol, 10mM Na₂HPO₄ anhidro). Then cells were sonicated and 20 μl of 50% β-mercaptoethanol - 50% Bromofenol blue were added. The protein concentration was determined using the Lowry assay. Proteins were resolved on SDS-polyacrylamide gels and transferred onto PVDF Membrane (Biorad). The blot was blocked with 5% milk powder in PBS1X with 0.1% Tween-20 for 60 minutes and incubated overnight with 1% milk powder in PBS1X with 0.1% Tween-20 with the following antibodies: anti-PARP-1(C2-10 mouse, ALEXIS, LA), anti-PTEN (Santa Cruz Biotechnology sc-7974), anti-BRCA1 (Santa Cruz Biotechnology sc-642), anti-RAD51 (Santa Cruz Biotechnology (H-92) sc-8349), anti-phosphoERK (Santa Cruz Biotechnology sc-7383), anti-ERK (Invitrogen. Carlsbad, CA 61-7400), anti-phospho H2AX (Millipore 05-636) and anti-BUBR1 (BD Bioscience. Erembodegem, Belgium). α-Tubulin (Sigma, St Louis MO) and β-Actin (Sigma, St Louis MO) were used as loading control. Bands were visualized with ECL, ECL-PLUS and ECL PRIME (Amersham Biosciences) and the pictures were taken with the imaging system ChemiDoc XRS System (BIO-RAD) and medical X-ray films (AGFA).