Sab1405848

Protein was extracted from cells using cell lysis solution with protease
inhibitors (Roche, 04693159001; Basle, Switzerland) and phosphorylase inhibitors
(Roche, 04906845001). Protein concentration was determined with a BCA protein
assay kit (Thermo Fisher Scientific, 23227, Massachusetts, USA). Equal amounts
of protein were fractionated on Tris-glycine SDS-polyacrylamide gels and
subjected to electrophoresis and transferred to NC membranes. Membranes were
blocked with TBS containing 5% (w/v) dry milk with 0.1% Tween 20, washed with
TBS containing 0.1% Tween 20 (TBST), and then incubated overnight at
4°C with specific antibodies against TIGAR (1:1,000; Abcam, ab37910;
Cambridge, UK), G6PD (1:1000; CST,#8866; Massachusetts, USA), Ckd5 (1:1000;
Abcam, ab40773), p-ATM (1:1000; Abcam, ab81292), ATM (1:1000; Abcam, ab78),
GAPDH (1:2000; Sigma, SAB1405848) in non-fat milk containing 0.1% NaN3. After
washing in TBST, membranes were incubated with fluorescent secondary antibodies
(1:10,000; Jackon ImmunoResearch, anti-rabbit, 711-035-152, anti-mouse,
715-035-150; West Grove, PA, USA) at room temperature for 1 h.
Immunoreactivity was detected using ODYSSEY INFRARED IMAGER (Li-COR Biosciences,
Nebraska, USA). The signal intensity of primary antibody binding was
quantitatively analyzed with Image J software (W.S. Rasband, Image J, NIH,
Bethesda, MD).

Western blotting was performed using antibodies directed against CD31 (1:1000; ab24590; Abcam) and GAPDH (Sigma-Aldrich; SAB1405848; 1:6000). GAPDH served as an internal control. The cells were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (Cell Signaling Technology) containing PMSF (1:100; v/v) (Cell Signaling Technology) for 30 min. The BCA Protein Assay Kit (Pierce, Thermo Scientific) was used to measure the total protein concentrations. Aliquots (40 μg) of total cellular protein were resolved by SDS-PAGE (10~12%), electrotransferred to PVDF membranes, and blocked with 5% skim milk (w/v) at room temperature for 1 h. The membranes were then incubated with primary antibodies on an orbital shaker at 4 °C overnight, and secondary antibodies (HRP-conjugated goat anti-mouse and HRP-conjugated goat anti-rabbit) were added and incubated for 1 h at room temperature. Protein-antibody complexes were then detected by chemiluminescence (Pierce ECL Western Blotting Substrate, Thermo, USA).