Rabbit anti chk1

Antibodies were obtained from the following sources: anti-RPA/p34 (Neomarkers MS-691-P0), anti-γ-H2AX (Millipore 05-636), rabbit anti-BLM (Eladad et al., 2005 (link)), rabbit anti-RNF4 (a gift from Dr. Jorma Palvimo), rabbit anti-CHK1 (Cell Signaling Technology 2345) at 1:400, rabbit anti-phospho-CHK1 (ser317) (Cell Signaling Technology 2344S) at 1:400, rat anti-HSC70 (Assay Design) at 1:45,000, anti-tubulin (Sigma T9026), anti-SUMO2 (Zhang et al., 2008 (link)), anti-Myc (Cell Signaling Technology 2276S), and anti-SENP6 (a gift from Dr. Mary Dasso). AlexaFluor-labeled secondary antibodies (A11029; A11035), were obtained from Invitrogen. HRP-labeled secondary antibodies were anti-mouse IgG (Cell Signaling Technology 7076S), anti-rat IgG (Jackson Labs), and anti-rabbit IgG (GE Healthcare NA934V). For DNA fiber assays, antibodies for detection of 5-iodo-2′-deoxyuridine (IdU) were mouse anti-IdU (BD) and for detection of 5-chloro-2′-deoxyuridine (CldU) were rat anti-CldU (Abcam); the secondary antibodies were anti-mouse Dylight 488 and anti-rat Dylight 649 (Jackson ImmunoResearch).

Medium and cells were harvested in PBS-containing 1 mM Na₃VO₄ and 1 mM NaF. Cells were pelleted before lysis in 50 mM Tris.HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% deoxycholate and 0.1% SDS. Samples were thawed on ice, centrifuged at 14,000 rpm for 20 min at 4 °C and supernatants quantified by BCA assay from Pierce (Leicestershire, UK). 30 μg total protein lysate was separated by reducing SDS-PAGE, transferred to PVDF (GE Healthcare, Bucks, UK) and blocked with 5% non-fat dry milk in TBS. The following primary antibodies were used: rabbit anti-HSP72 from Stressgen (Exeter, UK); rabbit anti-GAPDH, rabbit anti-ATR, rabbit anti-phospho-ATR (S428), rabbit anti-CHK1, rabbit anti-phospho-CHK1 (S345), rabbit anti-RAD51, rabbit anti-ATM, rabbit anti-phospho-ATM (S1981), rabbit anti-phospho-BRCA1 (S1524), rabbit anti-phospho-p53 (S15) and rabbit anti-phospho-H2Ax (S139) were purchased from Cell Signaling (MA, USA); rabbit anti-FANCA was purchased from Bethyl Laboratories (TX, USA). Secondary antibodies used were sheep anti-mouse IgG and donkey anti-rabbit IgG HRP from GE Healthcare (Buckinghamshire, UK). Chemiluminescent detection was carried out using immobilon western substrate from Millipore (East Midlands, UK). In vivo samples were processed using a Precellys®24 homogenizer from Bertin Technologies (Montigny, France).