Activated caspase 3

Paraffin sections from animals’ stomachs were deparaffinised and hydrated to distilled water. Sections were then blocked with 5% BSA solution in PBS for 1 hour at room temperature and then incubated with the appropriate primary antibody overnight at 4 °C. The primary antibody used was the anti-human Transthyretin (TTR) from DAKO (A000202) at a dilution of 1/500. The slides were then incubated with Invitrogen Alexa Fluor 555 fluorescence secondary antibody for 1hour at room temperature at 1/2,000 (anti-rabbit A-21428). Finally sections were washed with PBS and mounted using the DAKO Fluorescence Mounting Medium (S3023).
Further analysis was carried out using supplementary antibodies; Fas (Santa Cruz Biotechnology anti-rabbit sc-10231/1000) and activated Caspase-3 (Santa Cruz Biotechnology anti-goat sc-1225 1/500) to assess apoptosis, GRP78 (Santa Cruz Biotechnology anti-rabbit sc-13968 1/1000) to assess the endoplasmic reticulum stress response, and C5b-9 (EMD Millipore anti-rabbit 204903 1/4,000) to assess complement activation and Megalin (Santa Cruz Biotechnology anti-goat sc-16478 1/400). The appropriate secondary antibodies were used, anti-rabbit (Alexa Fluor A-21428 1/2000) and anti-goat (Alexa Fluor A-21432 1/2000). ECL was used to visualize the antibodies as previously explained.

The study was conducted in accordance with the guidelines in the Declaration of Helsinki. The human oral squamous cell carcinomas (OSCC) SCC9 cell line was purchased from the Institute of Biochemistry and Cell Biology (Shanghai, China). It was maintained in DMEM-F12 (Gibco, Shanghai, China), and supplemented with 10% fetal bovine serum and maintained at 37°C in a 5% CO₂ humidified incubator. Antibodies against survivin, PUMA, activated caspase-3, and β-actin were from Santa Cruz (Shanghai, China). YM155 were purchased from Selleck Chemicals (Shanghai, China). PUMA siRNA, caspase-3 siRNA, and control siRNA were purchased from Santa (Shanghai, China).

Cytoplasmic and nuclear proteins were isolated from lung tissue samples following the instructions of the KGI Nuclear and Cytoplasmic Protein Extraction kit (Nanjing KGI Biotechnology). Protein concentrations were determined using the BCA method (Beyotime Institute of Biotechnology, Haimen, China). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and were transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA). The membranes were incubated overnight at 4°C with rabbit polyclonal antibodies against TLR4, NF-κB p65, Nrf2, HO-1, and activated caspase-3 (Santa Cruz Biotechnology, Inc., Dallas, TX). The membranes were then incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Beyotime Institute of Biotechnology) at 37°C for 45 min. The enhanced chemiluminescence (ECL) imaging system was to visualize protein bands. β-actin was used as the internal control.