Anti caspase 3

Proteins were extracted from cells with Laemmli sample buffer (Biorad). Equal amounts of each extract were electrophoresed in 10% sodium dodecyl sulfate-polyacrylamide (SDS) gel electrophoresis, and then transferred into nitrocellulose membranes. Membranes were blocked for 1.5 hours with fat free milk 5%. After washing, membranes were blotted with primary antibodies overnight. We used a 1/500 dilution for the primary antibodies anti-ATP2C1, anti caspase-3, anti-PARP (Santa Cruz Biotechnology, Santa Cruz, Germany) and anti-GAPDH (Cell Signaling). Membranes were then blotted with corresponding secondary antibodies (1:5000 dilution) for 2 hours and developed prior to adding Luminol reagent (Santa Cruz Biotechnology, Santa Cruz, Germany). The density of the bands was then revealed using image J software. Relative quantification was carried out by normalizing the relative densities of the desired protein bands to the densities of their corresponding loading control band.

GC (PubChem CID: 161120) and PQ were obtained from Sigma-Aldrich (Sigma, MO, USA). Anti-Nrf2, anti-HO-1, anti-NQO-1, anti-GCLM, anti-ICAM-1, anti-VCAM-1, anti-iNOS, anti-IKK-β, anti-IκB-α, anti-NF-κB p65, anti-phosphorylated (p)-IκB-α, anti-Bcl-2, anti-Bcl-xl, anti-Bax, anti-Caspase-3, anti-Caspase-9, anti-GAPDH, anti-β-actin, anti-histone, and IgG-HRP antibodies were products of Santa Cruz Biotechnology (Santa Cruz, Texas, USA). BCA protein concentration assay kit, PVDF membranes, and SDS-PAGE gel preparation kit were purchased from Beyotime Institute of Biotechnology. ECL plus kit was obtained from Nanjing KeyGen Biotech Co., Ltd. (KeyGen, Nanjing, CN). TNF-α, IL-1β, and IL-6 ELISA kits were obtained from Abcam (Cambrige, UK). GSH, NADPH, SOD, CAT, MDA, CK, and LDH kits were products of Nanjing Jiancheng Engineering Institute (Nanjing, CN).

GTCs were cultured in 24-well plates and infected with B.suis.S2 or B.suis.S2-mCherry for 24 h. Immunofluorescent staining of caspase-3, GRP78, CHOP, phosphoIRE1α, IRE1α, and LC3 was performed. The GTCs were fixed in 4% paraformaldehyde for 30 min and then permeabilized for 15 min with 0.1% Triton X-100 in PBS, subsequently blocked for 1 h with 5% BSA in PBS at room temperature, and co-incubated with anti-caspase-3 (Santa Cruz, 1:50 dilution), anti-CHOP (Santa Cruz, 1:50 dilution), anti-GRP78 (Santa Cruz, 1:50 dilution), anti-phosphoIRE1α (Abcam, 1:500 dilution), anti-IRE1α (Santa Cruz, 1:50 dilution), or anti-LC3 (Sigma, 1:500 dilution) antibodies at 37°C for 2 h. After washing and incubation with an anti-rabbit secondary antibody (for CHOP, phosphoIRE1α, IRE1α, LC3, and caspase-3) (Invitrogen, A21206; 1:500 dilution) or an anti-goat secondary antibody (for GRP78) (Invitrogen, A21432; 1:500 dilution) at 37°C for 1 h, the nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI) for 3–5 min. The fluorescent signals were examined under a Nikon A1R si confocal microscope system.

The expressions levels of apoptosis and autophagy allied proteins were evaluated by western blotting. Mukonal treated (3.75, 7.5 and 15 µM) and untreated (controls) tumerous breast cells (MDA-MB-231 and SK-BR-3) were lysed with RIPA buffer for protein collection. Each lysate was subjected to bicinchoninic acid assay for quantification of proteins and 30 µg of proteins from each sample were run on 10% SDS-PAGE. Thereafter, proteins were transferred to PVDF membranes and these membranes were blocked with non-fat milk at room temperature for 1.5 h. blocked membranes were subjected to suitable primary antibodies of 1:1000 dilution anti-caspase-3, anti-PARP, anti-BAX, anti-Bcl-2, anti-LC3B-I, anti-LC3B-II and anti-Beclin-1 (Santa Cruz Biotechnology, Inc., Dallas, United States) overnight at 4 °C. Afterwards, secondary antibody treatment was instigated with 1:1,000 dilutions of horse radish peroxidase‑conjugated anti‑rabbit secondary antibody (Santa Cruz Biotechnology, Inc., Dallas, United States) for 1 h at room temperature. Finally, the protein bands were visualized using ECL Advanced Western Blot Detection kit (GE Healthcare Life Sciences, Sweden).

In the present study, β-SiC nanowires were synthesized by directly annealing the amorphous SiOC nanocomposites in Ar atmosphere [17 ]. High-sugar Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (USA). Penicillin-streptomycin was purchased from Sangon (Shanghai, China). WST-8 was purchased from Dojindo Laboratories (Japan). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2,7-dichlorodihydrofluorescein diacetate (DCF-DA), propidium iodide (PI), and Hoechst 33342 were purchased from Sigma-Aldrich Chemical Company. Annexin V-FITC Apoptosis Detection Kit I was purchased from BD Biosciences (USA). The primary anti-Bax, anti-Bcl-2, anti-caspase-3, and anti-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Cells were cultured and treated as described above and then lysed with ice-cold lysis buffer containing 50 mmol/L Tris-HCl, pH 7.4; 1% NP-40; 150 mmol/L NaCl; 1 mmol/L EDTA; 1 mmol/L phenylmethylsulphonyl fluoride; and complete proteinase inhibitor mixture (one tablet per 10 mL; Roche Molecular Biochemicals, Indianapolis, IN, USA). After protein content determination using a DC Protein Assay kit (Bio-Rad Laboratories, Hercules, CA, USA) and subsequently incubation with dilute solution (1 : 1000) of primary antibodies including anti-Bax, anti-Bcl-2, anti-caspase3, and anti-actin (Santa Cruz Biotechnology Inc, Santa Cruz, CA). The membranes were then exposed to the secondary antibodies, that is, alkaline phosphatase-labeled goat anti-rabbit immunoglobulin (Santa Cruz), at a dilution of 1 : 1000, followed by exposure to X-ray film [19 (link)]. The protein bands were detected using an enhanced chemiluminescence western blotting detection kit (Amersham, Little Chalfont, Buckinghamshire, UK).