Rabbit anti cleaved caspase 3 polyclonal antibody

Western blot analysis was used to detect expression of pro-apoptotic (AIF, Bax) and anti-apoptotic (Bcl-2, Bcl-xL) proteins as well as p-Akt and cleaved Caspase-3. Tissue samples from the ischemic cerebral hemispheres of all experimental and control groups were harvested at 6 (n = 6 per group) or 24 h (n = 6 per group) after reperfusion, as described previously by us⁴⁵ . PVDF membranes after protein transfer were incubated with primary antibodies including rabbit polyclonal anti-Phospho-Akt antibody (1:1000, Cell Signaling Technology), rabbit polyclonal anti-Bcl-2 antibody (1:200, Santa Cruz), mouse monoclonal anti-Bcl-xL antibody (1:200, Santa Cruz), mouse monoclonal anti-AIF antibody (1:4000, Santa Cruz), rabbit polyclonal anti-Bax antibody (1:500, Santa Cruz), and rabbit polyclonal anti-cleaved Caspase-3 antibody (1:1000, Cell Signaling Technology) at 4 °C for 24 h. Membranes were then incubated with the relevant secondary antibody (goat anti-mouse IgG, ZhongShanJinQiao; goat anti-rabbit IgG, ZhongShanJinQiao) for 1 h at room temperature. Equal protein loading was adjusted using β-actin (mouse polyclonal anti-β-actin antibody, Santa Cruz). An ECL system was used to detect immunoreactive bands by luminescence. Quantification of relative target protein expression was obtained using an image analysis program (Image J 1.48, National Institutes of Health, USA).

Cells were lysed in an RIPA lysis buffer (Millipore Corporation, Billerica, MA, USA) containing the protease inhibitor cocktail Complete Mini (Roche Diagnostics). Cell lysates were centrifuged at 15,000×g for 5 min, and then we collected the supernatant and measured the protein concentration. Ten micrograms of protein of each sample were electrophoresed on NuPAGE Bis–Tris Gels in NuPAGE MES SDS Running Buffer and transferred to PVDF blotting membranes (Thermo Fisher Scientific, Wilmington, DE, USA). Membranes were blotted with the following primary antibodies: rabbit polyclonal anti-bcl2 antibody (sc-492, Santa Cruz Biotechnology, Dallas, TX, USA), rabbit polyclonal anti-cleaved caspase3 antibody (#9661, Cell Signaling Technology, Danvers, MA, USA), and rabbit polyclonal anti-GAPDH antibody (GTX100118, GeneTex, Irvine, CA, USA) at 4 °C overnight and then with an anti-rabbit IgG, horseradish peroxidase-linked species-specific whole antibody (Amersham, Piscataway, NJ, USA) at room temperature for 1 h. Bound antibodies were then visualized using the ECL Western blot detection reagent (Amersham).

Proteins (40–50 µg) were resolved using denaturing 10 or 15% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked in 5% skim milk in Tris-buffered saline with 0.1% Tween 20 (TBST) and subsequently incubated overnight at 4°C with the following primary antibodies: goat anti-IGFBP3 polyclonal antibody (1∶2000, R&D Systems), rabbit anti-cleaved caspase 3 polyclonal antibody (1∶1000, Cell Signaling), and mouse anti-β-actin monoclonal antibody (1∶2000, Santa Cruz). After washing, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase for 1 h at room temperature. Chemiluminescence was detected using Super Signal West Dura substrate (Thermo Scientific) according to the manufacturer's protocol. Bands were visualized using a Luminescent Image analyser LAS-300 (General Electric) and quantified using Image Gauge software (Science Lab)

Genipin, wortmannin, rapamycin, and torkinib were purchased from Selleck (Shanghai, China). Lipofectamine 2000 and Opti-MEM were purchased from Invitrogen (Carlsbad, CA, USA). LPS from E. coli 0111:B4, ATP, nigericin, MSU, and z-VAD-fmk were purchased from InvivoGen (San Diego, CA, USA). Alum, flagellin and N-acetylcysteine (NAC) were purchased from Sigma (Shanghai, China). Caspase-1, ASC and IL-1β antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-UCP2 polyclonal antibody and GAPDH were purchased from Proteintech (Wuhan, China). Rabbit anti-LC3 polyclonal antibody was obtained from Abcam (Cambridge, MA, USA). Rabbit anti-cleaved caspase-3 polyclonal antibody was purchased from Cell Signaling (Beverly, MA). Mouse anti-β-tubulin monoclonal antibody was purchased from Sungene Biotech (Tianjin, China).

Immunohistochemical analyses were performed on REM134 tumor xenografts resected from mice injected once with VVTG17990 intratumorally at 10⁶ PFUs or intravenously at 10⁷ PFUs. Tumors were harvested 2 weeks after virus injection and fixed in 4% PFA for 1 h and overnight in 30% sucrose and OCT (coating medium for frozen tissue sections) embedded. To evaluate VACV infection, vascularization, and apoptosis of the tumor, samples were sectioned and stained using the following primary antibodies: rat anti-CD31 monoclonal antibody MC13.3 (dilution 1/500) (557355, BD Biosciences, San Jose, CA, USA), rabbit anti-VACV polyclonal antibody (dilution 1/1,000) (B65101R, Meridian Life Science, Memphis, TN, USA), and rabbit anti-cleaved caspase-3 polyclonal antibody (dilution 1/200) (9661, Cell Signaling Technology, Leiden, the Netherlands). In addition to the immunohistochemistry staining, slides were counterstained with DAPI to label nuclear DNA, and direct detection of GFP expression under fluorescence microscopy on tissue section was done.