Rad51

Antibodies for histone H4 (Millipore, 05858), H4K16ac (Millipore, 07329) SMARCAD1 (Santa Cruz Biotech, sc-162233, Novus Bio NB100–79835), γ-H2AX (Millipore, JBW301), Ku80 (Cell Signaling, 2180P), RAD51 (Abcam, ab213), BRCA1 (Santa CruzBiotech,sc-642), 53BP1 (Santa Cruz Biotech, sc-22760), CSB (Santa Cruz, 25371, 166042), GAPDH (Protein Tech, HRP-6004), were used for western blotting, immunoprecipitation and immunofluorescence. Antibodies used for the chromatin immunoprecipitation (ChIP) assay were histone H4 (Millipore, 05858), H4K16ac (Millipore, 07329), SMARCAD1 (Abnova, H00056916-BO1P), γ-H2AX (Millipore, JBW301), 53BP1 (Santa Cruz Biotech, sc-22760), BRCA1 (Abcam), Ku80 (Thermo Fisher,MA5–12933), RAD51(Abcam,ab176458), RAD51 (Abcam, ab176458), and RNA polymerase II (Abcam, ab817).

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.

Cellular protein was extracted using RIPA buffer (Beyotime) containing phenylmethylsulfonyl fluoride and phosphatase inhibitors. Equal amounts of extracted cellular protein were resolved on 6–12% SDS-PAGE-denaturing gels and transferred electrophoretically onto polyvinylidene difluoride membranes. The membranes were blocked with 5% no-fat milk for 2 h at room temperature. The bands were incubated with the following diluted primary antibodies: p-AMPK, AMPK, p-mTOR, mTOR, Beclin-1, LC3B, γH2A.X (Cell Signaling, MA, USA, 1 : 1000), P21, P16 (Abcam, Cambridge, UK, 1 : 1000), Rad51 (Abcam, UK, 1 : 5000), and β-actin (Boster, Wuhan, China, 1 : 5000) overnight at 4°C. After washing three times with TBST, the membranes were then incubated for 2 h at room temperature with goat anti-rabbit or anti-mouse secondary antibodies (Boster, Wuhan, China, 1 : 5000). The excessive secondary antibody was washed off three times with TBST, and the bands were visualized using the Bio-Rad imaging system with the ECL imaging kit (Millipore Corporation). The intensity of the images was quantified by using ImageJ software.