Chk1 s317

HCT-116 cells were grown in T25 flasks for 24 h (exponential growth), treated with anti cancer drugs (as indicated in figure legends) and harvested by scraping 24 h post treatment. Cells were centrifuged at 1500 rpm for 5 min and pellets washed in cold PBS. Pellets were dissolved in lysis buffer as previously described (Davidson et al., 2012b (link), 2013 (link)). Cell lysates were separated by SDS-PAGE, transferred to nitrocellulose membranes under the appropriate conditions, and blotted for the following antigens: total Chk1 (Santa Cruz, sc-8408), DNA-PK (Upstate, 05-423) and Rad51 (Upstate), phosphorylated antibodies; Chk1 (S317) (Cell Signaling, 2344), DNA-PK (S2056) (Abcam, ab18192) and Rad51 (T309) (Abcam) and β-actin (Santa Cruz, sc-1616). Chk1, DNA-PK and Rad51 levels were normalized to β-actin and phosphorylated-Chk1, -DNA-PK, and -Rad51 were normalized to total-Chk1, total-DNA-PK and total-Rad51 respectively. Each experiment was repeated at least 3 times. Blots were quantified using ImageJ image analysis software.

Cells were harvested in UTB lysis buffer (9 M Urea, 75 mM Tris-HCl pH 7.5, 0.15 M β-mercaptoethanol). Blots were visualised using the Odyssey Infrared imaging system. Antibodies used were SETX, KAP1 (Bethyl); GRP78, HIF1α, (ΒD Biosciences); ATM-S1981, RPA-S4/8, β-Tubulin (Abcam); ATM, RPA, KAP1-S824, p53-S15, H3K9me2, H3K4me2, H3K9Ac, H3-S10, H3, H3K36me3, Chk1-S317, PERK (Cell Signaling); p53, β-actin, RNase H1, Chk1 (Santa Cruz); and H3K9me3, γH2AX, H2AX (Millipore).

The antibodies used were ATR-T1989 (Millipore), Chk1-S317, Chk1-S345, KAP1-S824 (Cell Signaling Technology), KAP1-S473 (Biolegend), KAP1 (Bethyl/Universal Biologicals Cambridge), Chk1, ATR, β -actin (Santa Cruz Biotechnology), HIF-2α (Novus Biologicals), HIF-1α(BD Biosciences) and GAPDH (Stratech Scientific).