Parp 46d11 rabbit mab

Cells were lysed with RIPA lysis buffer (sc-24948, Santa Cruz Biotechnology, Inc., Dallas, TX) according to the protocol supplied. Whole cell lysates (30 μg) were separated by SDS-PAGE, transferred to nitrocellulose through Trans-Blot Turbo RTA Mini Nitrocellulose Transfer Kit (Bio-Rad, Hercules, CA, USA). Membranes were subjected to immunoblot analyses using primary antibodies against phosphorylated RB (Ser780) #8180 1:1,000 (Cell Signaling Technology, Danvers, MA), phosphorylated RB (Ser807/811) #8516 1:1,000 (Cell Signaling Technology), RB (IF-8) #sc102 1:1,000 (Santa Cruz Biotechnology, Inc.), Erα (F-10) #sc-8002 1:200 (Santa Cruz Biotechnology, Inc.), PLK1 (208G4) #4513 (Cell Signaling Technology) 1:1,000, β-actin (13E5) rabbit mAb #4970 (Cell Signaling Technology) 1:5,000, PARP (46d11) rabbit mAb #9532 (Cell Signaling Technology), GAPDH #sc-32233 1:10,000 (Santa Cruz Biotechnology, Inc.), HRP-conjugated anti-rabbit and anti-mouse were used as secondary antibodies (Bio-Rad). Immunoreactive proteins were visualized by enhanced chemiluminescence using SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA). Membranes were cut horizontally to probe with multiple antibodies. Films were imaged using Brother MFCL2710DW (Brother) at 300 dpi.

The cell lysates were homogenized in mammalian protein extraction buffer (Sigma). The resulting lysates were centrifuged at 12,000 rpm, and 4°C for 15 min, and the protein contents were measured using a protein-assay dye reagent concentrate (Bio-Rad Laboratories, Hercules, CA, USA). Subsequently, 10–15% sodium dodecyl sulphate-polyacrylamide gel electrophoresis was performed, followed by transfer onto nitrocellulose blotting membranes (Amersham, GE Healthcare Life Science, Germany). The antibodies used for immunoblotting are as follows: Bax rabbit mAb (1:1,000; Cell Signaling Technology), Bcl2 (D17C4) rabbit mAb (1:1,000; Cell Signaling Technology), inducible nitric oxide synthase (iNOS; NOS2) (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), AMPKα (D63G4) rabbit mAb (1:1,000; Cell Signaling Technology), Phospho-AMPKα (Thr172) (D4D6D) rabbit mAb (1:1,000; Cell Signaling Technology), caspase-3 rabbit mAb (1:1,000; Cell Signaling Technology), cleaved caspase-3 (Asp175) (5A1E) rabbit mAb (1:1,000; Cell Signaling Technology), PARP (46D11) rabbit mAb (1:1,000; Cell Signaling Technology), and β-actin (13E5) rabbit mAb (1:2,500; Cell Signaling Technology). The protein bands were visualized using an enhanced chemiluminescence method with an ELC kit (Millipore, Billerica, MA, USA) and were quantified using Quantity 1 v.4.6.7 (Bio-Rad).