Anti phospho atm s1981

Cells were fixed for 15 min. with 4% paraformaldehyde in PBS and permeabilized with PBST solution (0.5% Triton X‐100 in PBS) for 30 min. After blocking with 5% bovine serum albumin in PBST solution for 1 hr, cells were incubated with the anti‐phospho H2AX (S139) (05‐636; Millipore, Temecula, CA, USA), anti‐53BP1 (sc22760; Santa Cruz Biotechnology), anti‐phospho ATM (S1981) (ab1888; Abcam) antibodies for overnight at 4°C respectively. Antigen was detected with the secondary antibodies conjugated to FITC (F0382; Sigma‐Aldrich) and TRITC (T5393; Sigma‐Aldrich). Cells were coverslipped using VECTASHIELD mounting media plus DAPI (H‐1200; Vector Laboratories, Burlingame, CA, USA). Images were acquired with a confocal microscope (Leica TCS SPE; Leica Microsystems GmbH, Wetzlar, Germany). Percentages of γH2AX foci positive cells were counted in five random high power fields.

For western blotting, 18 µg of protein was loaded onto a 4–15% sodium dodecyl-sulfate polyacrylamide gel for electrophoretic separation (Cat. No. 4,561,085, Bio-Rad, Hercules, CA, USA). The primary antibodies used in this study are as follows: anti-ATM (1:2000; Cat. No. ab32420, Abcam, Cambridge, UK); anti-phospho S1981-ATM (1:50 000; Cat. No. ab81292, Abcam); anti-HIF1α (1:1000; Cat. No. ab51608, Abcam); anti-phospho-HIF1α (1:1000; Cat. No. AF0062, Funakoshi, Tokyo, Japan); anti-Met (1:1000; Cat. No. 3127, Cell Signaling Technology Danvers, MA, USA); anti-Netin1 (1:1000; Cat. No. ab126729, Abcam); Anti-Apoptosis Sampler Kit (anti-BCL-2: 1:1000; anti-BAD: 1:1000; Cat. No. E051004, Funakoshi). Anti-β-actin antibody (1:10 000; Cat. No. 66009-1, Proteintech, Tokyo, Japan) was used as a standardized internal reference to compare the target protein expression levels. Finally, immunoreactive bands were detected using the ChemiDoc Imaging System (Bio-Rad) and analyzed using ImageJ software (NIH, Bethesda MD, USA).