Ab2905

Immunoblotting was performed by using the proteins immunoprecipitated by anti-ATR (ab2905, Abcam), anti-RAD18 (ab188235, Abcam), anti-human gamma H2A.X (phosphor S139) (ab81299, Abcam) and anti-RAD51C (A302-645A, Bethyl Laboratories) antibodies. Briefly, the indicated cell lysates were separated by SDS-PAGE, and were transferred onto a PVDF membrane. The membrane was then preincubated in 10% skimmed milk at 4°C for 1 h. Then, the human recombinant DDUP protein (Abclonal, China) was added and further incubated for 18 h at 4°C. The membrane was then washed six times with TBST buffer and subjected to immunoblotting analysis by anti-DDUP antibody (Sino Biological, China).

100 μg protein was loaded onto 10% Mini-PROTEAN TGX precast gels (Bio-Rad; 456-1043) and run at 150 V for ∼40 min in Tris/glycine/SDS buffer (Bio-Rad; 1610732). Proteins were transferred onto PVDF membrane at 100 V for ∼35 min in transfer buffer (10 mmol/l CAPS, 10% methanol, pH 10.5). Membrane was blocked for 1 h with 5% non-fat milk in TBS–Tween. Proteins were probed overnight at 4°C with anti-ERCC1 (Abcam; ab76236; 1:1000), anti-XPF (Santa Cruz; sc-136153; 1:1000), or for 1 h at room temperature with anti-β-actin (Sigma-Aldrich; A5441; 1:100 000) or loading control α-tubulin (Sigma-Aldrich; T5168; 1:100 000) in antibody dilution buffer (3% w/v bovine serum albumin (BSA), 0.2% v/v sodium azide in PBS–Tween). For the ATR western blot, similar reactions were conducted with ATR antibody (Abcam; ab2905; 1:500). Excess antibody was removed by washing three times with PBS–Tween and the membrane was subsequently probed with goat anti-mouse or goat anti-rabbit secondary antibodies (Bio-Rad; 172-1011 and 172-1019; 1:2000) for 45 min at room temperature. Excess secondary antibody was removed by washing three times with PBS–Tween. PDX tumor tissue as well as resected lung tumor tissue for ERCC1 western blot assessment was obtained from the Karmanos Cancer Institute biorepository and PDX core with patient information de-identified and thus, IRB exempt.