Total chk1

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.

Ru1 was prepared using methods described by Bolger, et al.61 All NMR, mass spectroscopy and elemental analysis were in agreement with published data. Unless stated otherwise, all other chemicals were obtained from Sigma. Antibodies: p-Chk1 (Ser345) and p-Chk2 (Thr68) (Cell Signaling), γH2AX (Millipore), total Chk1 (Santa Cruz), total Chk2, α-tubulin (both Abcam) and β-actin (Sigma). AlexaFluor488-conjugated anti-mouse secondary antibodies were from Cell Signaling. DTPA-hEGF was prepared as reported previously.62 (link)