Pchk1 s296

Proteins were isolated and their differential expressions were analyzed by Western blot, as described previously [21 (link),22 (link)]. Briefly, the cells were lysed using RIPA buffer (50 mM Tris HCl at pH 7.4, 150 mM NaCl, 1% Triton X-100 or NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, and 10 mM NaF freshly supplemented with protease and phosphatase inhibitors). Protein concentrations were quantified using a Bradford assay (Bio-Rad), and the samples were normalized for equal loading. The samples were then heated to 100 °C for 10 min in the presence of 6× Laemmli buffer (Boston BioProducts; Milford, MA, USA). The proteins were resolved by SDS-PAGE and their expression was analyzed following immunoblotting using specific antibodies. The following antibodies were used in this study: FANCD2 (R&D MAB93691), pCHK2-T68 (Invitrogen PA5-104715), pRPA 32-S33 (Cell Signaling 10148s), pCHK1-S296 (Cell Signaling 90178s), pH2AX-S139 (Millipore 05-636), GAPDH (Santa Cruz 32233), and Vinculin (Cell Signaling 13901S).

Cells were lysed with a buffer containing 67 mM Tris-HCl (pH 6.8) and 2% SDS. Denatured proteins were then resolved by SDS-PAGE and transferred to nitrocellulose membranes as previously described [50 (link)]. Proteins were stained with Ponceau S (0.1% Ponceau reagent [Sigma, P3504] with 5% acetic acid). Incubation with primary antibodies was conducted overnight at 4°C. Antibodies against the indicated proteins were used as follows: RAD51 (Santa Cruz Biotechnology, Dallas, TX, USA, H-92, sc-8349; 1/200), Cyclin A2 (Santa Cruz Biotechnology, H-432 sc-751; 1/500), P-Chk1 S296 (Cell Signaling, Danvers, MA, USA, #2349; 1/1000), Lamin B (Santa Cruz Biotechnology, M-20 sc-6217; 1/200), GAP120 (Santa Cruz Biotechnology, sc-63; 1/100), CDK4 (Santa Cruz Biotechnology, sc-749; 1/500), Polymerase α (Santa Cruz Biotechnology, sc-5921; 1/200), RAD51 (Santa Cruz Biotechnology, sc-8349; 1/200). Proteins were visualized using EZ-ECL Kit (Biological Industries). Signals were detected using imaging. Quantification was done by densitometry using Image Lab^™ Software (Bio-Rad, Hercules, CA, USA, #1709690). Original uncropped and unadjusted images are included in the S1 Raw images (note that some of the membranes were cropped before incubated with the antibodies in order to be able to evaluate different proteins in a single sample).

Human TNBC cell lines MDAMB231, MDAMB453, and MDAMB468 were purchased from ATCC, Manassas, VA. All three cell lines were cultured in Dulbecco’s modified Eagle medium (Corning, Manassas, VA), supplemented with 10% fetal bovine serum (Omega Scientific Inc., Tarzana, CA) and 1% penicillin-streptomycin (50 U/mL, 50 μg/mL, Invitrogen, Eugene, OR). Prexasertib (Sellechem, Houston, TX), olaparib (Sellechem, Houston, TX), epoxomicin (Sigma, St. Louis, MO), and MG132 (Sellechem, Houston, TX) were dissolved in DMSO and used at the specified concentrations and times as indicated. The following primary antibodies were used for western blotting: RAD51 (Santa Cruz Biotechnology, Santa Cruz, CA), BRCA1 (Santa Cruz Biotechnology, Santa Cruz, CA), γH2AX (Millipore, Billerica, MA), pCHK1 S296 (Cell Signaling, Danvers, MA), CHK1 (Santa Cruz Biotechnology, Santa Cruz, CA), and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA).