Rabbit anti h2ax

Whole cell lysates were prepared by boiling cell pellets for 10 min in SDS lysis buffer [2% SDS, 10% Glycerol, 62 mM Tris-HCl, pH 6.8 and a complete mini-protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN)]. After protein quantification with Bio-Rad Dc Protein Assay (Bio-Rad Laboratories, Hercules, CA), equal amounts of proteins were loaded, separated on a polyacrylamide gel, and transferred to a nitrocellulose membrane. Protein bands were immuno-detected with appropriate antibodies, e.g., rabbit-anti-XPC [47 (link)], mouse-anti-E-Cadherin (BD Transduction Laboratories, San Jose, CA, Cat. No. 610181, 1:1000), rabbit-anti-Snail (Cell Signaling, Danvers, MA, Cat. No. 3879, 1:1000), rabbit-anti-pERK1/2 (Cell Signaling, Cat. No. 9101, 1:1000), rabbit-anti-ERK2 (Cell Signaling, Cat. No. 9108, 1:1000), mouse-anti-γH2AX (Millipore, Billerica, MA, Cat. No. 05-636, 1:1000), rabbit-anti-H2AX (Cell Signaling, Cat. No. 7631, 1:1000), mouse-anti-Actin (Santa Cruz Biotechnology, Dallas, TX, Cat. No. sc-47778, 1:1000), and goat-anti-Lamin B (Santa Cruz Biotechnology, Cat. No. sc-6216, 1:1000).

Proteins were separated by electrophoresis in 12% and 6% polyacrylamide gels according to molecular weight. Then, they were transferred to 0.2 μm nitrocellulose membranes (Bio-Rad) using transfer buffer (25 mM Tris, 190 mM glycine and 20% methanol). Membranes were blocked for 60 min at 37 °C with 3% bovine serum albumin (BSA) in TBST-1X (150 mM NaCl, 20 mM Tris, 0.1% Tween-20, at pH 7.5) and incubated overnight at 4 °C with mouse anti-tubulin (1:2,000 Cell Signaling), mouse anti-GOLPH3 (1:1,000 Santa Cruz), rabbit anti-H2A.X (1:500, Cell Signaling), mouse anti-TGN38 (1:500 Santa Cruz), rabbit anti-phospho AKT1 (1:1,000, Cell Signaling), and rabbit anti-phospho mTOR (1:1,000 Cell Signaling) antibodies. After washing, membranes were incubated with anti-rabbit or anti-mouse secondary antibodies (1:2,500) (Jackson ImmunoResearch). Chemiluminiscent detection of immunodetected bands was performed using the ECL Western blot detection reagent (Amersham).

Cells harvested, pelleted at 1,500 rpm at 4°C for 5 min and lysed with whole cell lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, and cocktail protease inhibitors). Protein concentration was determined using the Bradford assay (Sigma). Protein bands were separated on a NuPAGE 4–12% Bis-Tris Gel (Invitrogen). Proteins were electrotransferred onto a nitrocellulose membrane in 1X NuPAGE transfer buffer. After blocking with 5% skimmed milk solution in 1% Tris-Buffered saline with Tween 20 (TBST) buffer, the membranes were immunoblotted with mouse anti-Flag antibody (Sigma, #F3165, 1:1000), rabbit anti-TDP-43 (Proteintech, #10782-2-AP, 1:1000), mouse anti-phospho-Histone H2.AX (S139) (EMD Millipore, #16-193, 1:1000), rabbit anti-H2.AX (Cell Signaling Tech, #2595, 1:1000), rabbit anti-phospho-ATM (S1981) (Abcam, #ab81292, 1:800), rabbit anti-ATM (Abcam, #ab32420, 1:1000), rabbit anti-phospho-53BP1 (S1778) (Cell Signaling Tech, #2675, 1:1000), rabbit anti-53BP1 (Cell Signaling Tech, #4937, 1:1000), and mouse anti-β-actin (Proteintech, #66009-1-Ig, 1:5000) antibodies. Protein bands were visualized by probing with corresponding HRP-conjugated secondary antibodies and developed with enhanced chemiluminescence reagent in Odyssey (LI-COR).