Rabbit anti caspase 3 antibody

SW10 mouse Schwann cells, L929 mouse fibroblasts, J774A.1 mouse macrophage, C2C12 mouse myoblast, Neuro-2a mouse neuroblast, sNF96.2 human Schwann cells and HUVEC human endothelial cells were cultured for 24 hrs. Synthetic mycolactone A/B with a final concentration of 300 ng/ml, 30 ng/ml, or 3 ng/ml was then added and the cells were incubated further. Floating and adhered cells were collected at 12, 24 and 48 hrs time points. Ethanol similarly diluted with culture media to a final ethanol concentration of 300 ng/ml was used as the negative control. In addition, actinomycin-D (Sigma) diluted with culture medium for 24 hrs was used as the positive control. Following the bicinchoninic acid assay (BCA assay), Western blot analysis was performed using rabbit anti-cleaved caspase-3 (Cell Signaling #9661), rabbit anti-caspase-3 antibody (Cell Signaling #9662), mouse monoclonal anti-histone H2A.XS139ph (phospho Ser139) antibody (GENETEX, Inc. GT2311), and mouse monoclonal anti-α-tubulin (Sigma T-9026) as an internal control. Horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (7076) and goat anti-rabbit IgG (7074), purchased from Cell Signaling, were used as secondary antibodies. Immunoreactive bands were visualized using a chemiluminescence reagent Immuno Star LD (Wako).

Human ovarian adenocarcinoma cells (SKOV-3) were purchased from American Type Culture Collection (Manassas, VA, USA). RES was purchased from MilliporeSigma (Burlington, MA, USA). MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was purchased from MilliporeSigma. Guava^® Cell Cycle Reagent was purchased from Luminex Corporate (Austin, TX, USA). Annexin V was purchased from ImmunoTools (Friesoythe, Niedersachsen, Germany). Propidium iodide was purchased from MilliporeSigma. Rabbit anti-phospho p38 antibody, rabbit anti p38 antibody, rabbit anti-phospho ERK1/2 antibody, mouse anti-ERK1/2 antibody, rabbit anti-phospho JNK antibody, rabbit anti-JNK antibody, rabbit anti-phospho AKT (Ser473) antibody, mouse anti-AKT antibody, rabbit anti-cyclin A2 antibody, rabbit anti-cyclin B1 antibody, rabbit anti-cyclin E1 antibody, rabbit anti-caspase-9 antibody, rabbit anti-caspase-3 antibody, rabbit anti-PARP antibody, mouse anti-β-actin antibody, and DAPI (4, 6-diamidino-2-phenylindole, dihydrochloride) were purchased from Cell Signaling Technology (Danvers, MA, USA). Goat anti-mouse IgG-IRDye^®800CW and goat anti-rabbit IgG-IRDye^®680RT were purchased from Li-COR Biosciences (Lincoln, NE, USA). Goat anti-rabbit conjugated with Alexa488 and Goat anti-mouse conjugated with Alexa594 were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

The TPs were meticulously isolated and then placed into lysis buffer containing cocktails of protease and phosphatase inhibitor. There were 4 samples in each group, and each sample consisted of TPs from both upper eyelids of the same mouse. The same amount of protein was loaded on 12% SDS-PAGE gels for electrophoretic separation, followed by being electronically transferred to a PVDF membrane. After blocking with 5% skimmed milk, the membrane was incubated with rabbit anti-caspase-3 antibody (1:1,000; 9662; Cell Signaling Technology, Danvers, MA, United States), rabbit anti- nuclear factor kappa B (NF-κB) antibody (1:1,000; 8242; Cell Signaling Technology), rabbit anti-phospho (p)-NF-κB antibody (1:1,000; 3033; Cell Signaling Technology), and mouse anti-β-actin antibody (1:1,000; AA128; Beyotime, Shanghai, China) at 4°C overnight. Then, the blots were incubated with HRP-labeled goat anti-mouse IgG (1:1,000; A0216; Beyotime) or anti-rabbit IgG (1:1,500; 7074; Cell Signaling Technology) for 1 h at room temperature. BeyoECL Star (P0018AS; Beyotime) was used to visualize the specific bands which were imaged using a chemiluminescence imaging system (GeneGnome XRQ; Syngene, Cambridge, United Kingdom). Staining intensities were quantified with ImageJ 1.52a software.