Cleaved parp antibody

IHC for pHistone H3 and cleaved PARP were conducted on 5 μm tissue sections from paraffin-embedded tumor as described previously using the EnVision + Single Reagents HRP-Rabbit (Dako) and REAL substrate buffer (REAL DAB + chromogen, Dako) [54 (link)]. The primary antibodies and dilutions are as follows: pHistone H3 (Ser 10) antibody (1:200; Cat. no. 06-570; Millipore), and cleaved PARP antibody (1:200, Cat. no. 9541; Cell Signaling).

Z-LIG with purity more than 98 % was purchased from Chengdu Must Bio-Technology Co, Ltd (Chengdu, China) and stored in −80 °C before use. The antibodies against BRCA1, ERα, HDAC1, HDAC2, HDAC4/5/7, Acetyl-Histone3(lys9/14), IFI16, and MTA1 were purchased from Santa Cruz Biotechnology (CA, USA). Acetyl-p53, pro-PARP and cleaved PARP antibody were obtained from Cell Signaling Technology (Boston, MA, USA). The antibodies against Cyclin A, Cyclin E, CDK1, CDK2, p53, Histone 3 were obtained from Wanlei Biotechnology (Shenyang, China). The antibodies against caspase 3, cleaved caspase 3, p21 and p27 were purchased from Proteintech Group Inc (Wuhan, China). The antibodies against β-actin and rabbit IgG were obtained from Sigma-Aldrich (St. Louis, MO, USA). Other chemicals were obtained from Sigma-Aldrich, unless indicated otherwise.

FACS-sorted cells were homogenized in lysis buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, and 1 μg/ml leupeptin (Cell Signaling Technology) supplemented with 1% Halt protease inhibitor cocktail (Thermo Fisher Scientific). Protein concentrations were then quantified using a Bio-Rad protein assay kit (Bio-Rad Laboratories). A total of 15 µg of each protein lysate from mCherry-positive cells of the Tg(lck:IRF4) fish were mixed with 4× Laemmli sample buffer (Bio-Rad Laboratories) and β-mercaptoethanol (Sigma-Aldrich) in appropriate proportions before being incubated at 99 °C for 5 min. The samples were then separated on SDS-PAGE gels and transferred to PVDF membranes (Millipore) at 400 mA for 1.5 h, blocked with 3% skim milk (Nacalai Tesque) in Tris-buffered saline with 0.1% Tween and probed with IRF4 antibody (1:1000; Santa Cruz), cleaved PARP antibody (1: 1000; Cell Signaling Technology) and GAPDH-HRP antibody (1: 1000; Santa Cruz). Secondary detection was performed with HRP-linked antirabbit IgG (Cell Signaling Technology) and ECL™ Western blotting detection reagents (Fisher Scientific). Western blot images were captured using an ImageQuant LAS500 chemiluminescent image analyzer (GE Healthcare).