Parp rabbit polyclonal

The drugs were dissolved in DMSO at the final concentration of 10 mM. Imatinib was purchased from Cayman Chemical (Michigan, USA) and used in the range of 5–25 µM; Gemcitabine was obtained from Sigma Aldrich Corp. (St Louis, MO, USA) and used in the range of 1–10 µM; Cisplatin, kindly donated by Prof. Justin Stebbing (Imperial College, London), was used in the range 1–25 µM. The following antibodies were used: MSLN mouse monoclonal (Santa Cruz); β-actin mouse monoclonal (Abcam), p53 mouse monoclonal (Santa Cruz); pERK, mouse polyclonal (Abcam); PARP rabbit polyclonal (Cell Signaling); pAKT rabbit polyclonal (Abcam); ERK1-2 rabbit polyclonal (Abcam); Secondary HRP (horseradish peroxidase)-conjugated goat anti-rabbit IgG and goat antimouse IgG antibodies were from GE Healthcare. The expression plasmid pcDNA3.1 encoding for MSLN (aa 360-2230) was kindly donated by Dr. Uehara (Kansai Medical University, Japan); the empty vector pcDNA3.1, employed as control, was donated by Dr. Giamas (Imperial College, London).

Cells were incubated in lysis buffer (1 % Triton X-100, 50 mM HEPES, 150 mM NaCl, 1 mM MgCl_2, 1 mM EGTA, 10 % glycerol, and protease inhibitor cocktail) for 1 h at 4 °C. Cell lysates were harvested by scraping and centrifuged at 14,000 rpm for 10 min at 4 °C. Normalized samples (using the BCA assay (Fisher Scientific, Pittsburgh, PA)) were run on SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes for western blotting. Bound antibody was detected using enhanced chemiluminescence reagent followed by exposure to film. Primary antibodies were used at the following dilutions and obtained from the following sources: Ago2 rabbit monoclonal (#2897, 1:500), caspase 2 mouse monoclonal (1:1000), caspase 3 rabbit monoclonal (1:1000), caspase 8 mouse monoclonal (1:1000), and caspase 9 mouse monoclonal (1:1000) (Initiator caspases sampler kit #12675), Drp1 rabbit monoclonal (#8570, 1:1000), GAPDH rabbit monoclonal (#2118, 1:5000), LC3B rabbit polyclonal (#2775, 1:1000), pan-actin rabbit polyclonal (#4968, 1:1000), PARP rabbit polyclonal (#9542, 1:1000), and PINK1 rabbit monoclonal (#6946, 1:500) antibodies were obtained from Cell Signaling Technology (Danvers, MA). ATG7 rabbit polyclonal antibody (PM039, 1:1000) was obtained from MBL International Corporation (Woburn, MA).

As previously described (24 (link), 25 (link)), proteins were extracted and normalized to at least 1500μg/ml. Proteins were subsequently analyzed on SDS-PAGE gels (8% or 10%, as appropriate) and transferred to polyvinylidene difluoride (PVDF) membranes for western blotting (24 (link), 25 (link)).
Primary antibodies that were utilized in this study are: SV40 LTAg mouse monoclonal (#554149, 1:1000, BD Biosciences), DNMT1 rabbit polyclonal (#5032, 1:1000, Cell Signaling Technology), HA-11 mouse monoclonal (#14921901, 1:500, Covance/Lab Corporation), CD71 mouse monoclonal (#sc-51829, 1:250, Santa Cruz Biotechnology), FTH1 rabbit polyclonal (#3998, 1:500, Cell Signaling Technology), pan-Actin rabbit polyclonal (#4968, 1:1000, Cell Signaling Technology), PARP rabbit polyclonal (#9542, 1:1000, Cell Signaling Technology), PAX-8 rabbit polyclonal (#10336-1-AP, 1:1000, Proteintech), Claudin-1 rabbit polyclonal (#187362, 1:10,000, Invitrogen), BAK rabbit monoclonal (#12105, 1: 1000, Cell Signaling Technology), Bcl-2 rabbit monoclonal (#2570, 1:1000, Cell Signaling Technology), Occludin-1 mouse monoclonal (#611090, 1:250, BD Biosciences), Cyclin D1 rabbit polyclonal (#sc-718, 1:1000, Santa Cruz Biotechnology), and Cyclin E mouse monoclonal (#sc-247, 1:500, Santa Cruz Biotechnology).