Horseradish peroxidase hrp conjugated goat anti mouse igg sc 2005

HeLa cells were provided by Riken (Tsukuba, Japan) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 μg/ml), in a humidified incubator with 5% CO₂ at 37 °C. 3-Aminobenzamide (3AB) was obtained from Tokyo Chemical Industry (Tokyo, Japan). Olaparib was purchased from ChemScene (NJ, USA), emetine hydrochloride from Cayman Chemical (No. 21048; MI, USA), and PDD00017273 [37 (link)] from Tocris Bioscience (MN, USA), and PD0325901 from ChemScene. The following antibodies were obtained from Santa Cruz Biotechnology (TX, USA): mouse anti-human PARP1 IgG (sc-8007), mouse anti-human phosphorylated ERK IgG (sc-7383), mouse anti-human ERK2 IgG (sc-1647), horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (sc-2005), and HRP-conjugated goat anti-rabbit IgG (sc-2004). Rabbit anti-human PARG IgG (ab16060) was purchased from Abcam (Cambridge, UK). Anti-PAR (10H, IgG3 kappa), secreted from hybridoma cells [38 (link)], was purified using a Protein A Sepharose Fast Flow column (GE Healthcare, IL, USA). Anti-PAR polyclonal antibodies were produced in rabbits by injecting PAR mixed with methylated BSA in our laboratory [39 (link),40 ]; the antibodies were purified with a Protein A Sepharose Fast Flow column.

The level of angiogenesis biomarker CD31 was measured by immunohistochemistry according to the previous report [23 (link)]. Briefly, the tissue sections were deparaffinized and rehydrated. Antigen retrieval was then performed followed by blocking of endogenous peroxidase through incubation in 3% hydrogen peroxide for 20 min at 37°C. After being blocked by incubating the tissue sections with 4% goat serum for 45 min, the sections were incubated with monoclonal mouse antirat CD31 antibody (NB100-64796, Novus Biologicals, USA, with 1 : 50 dilution) overnight at 4°C, followed by incubation with horseradish peroxidase- (HRP-) conjugated goat anti mouse IgG (sc-2005; Santa Cruz Biotechnology Inc., USA, 1 : 70 dilution) for 45 min at 37°C. Slides were stained using aminoethylcarbazole (AEC) chromogen substrate (AEC Staining Kit; Sigma-Aldrich) for 10 min, and the graphics were observed using the semiautomated computerized ImageJ software (Rasband; NIH, USA).

The following primary antibodies were used: anti-Kv4.3 (ab99045, for Western blot) and anti-Kv2.2 (ab10651) were acquired from Abcam (Hong Kong, China). Anti-Kv2.2 (sc-292489, for immunoprecipitation) and anti-p-Ser (sc-80514) antibodies were acquired from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-p-Ther antibody (#9386) was obtained from cell signaling technology (City and country of the company). The secondary antibody DyLight488 conjugated goat anti-mouse IgG (16117031711) was bought from ImmunoReagents, Inc. (Raleigh, NC, USA). The secondary antibody horseradish peroxidase (HRP) conjugated goat anti-mouse IgG (sc-2005) was bought from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Tetraethylammonium chloride (TEA), 4-Aminopyridine (4-AP) and other chemicals were all acquired from Sigma unless indicated otherwise.