Horseradish peroxidase conjugated goat anti rabbit antibody

Total proteins were extracted from treated cells using a radioimmunoprecipitation assay (RIPA) buffer (Invitrogen) for 30 min on ice. The suspension was centrifuged at 14,000 g for 10 min at 4 ℃; then, the total protein concentration in the supernatant was estimated using the BCA assay. Equal amounts of protein from different cultures were denatured by boiling at 100 ℃ for 5 min; then, 50 μg was separated on a Tris-Tricine gel at 70 V for 3 h. Fractionated proteins were transferred onto a PVDF membrane at 120 mA for 2 h, which was blocked using 5% nonfat milk in TBS-Tween. Membranes were incubated overnight at 4 ℃ with primary antibodies (all diluted 1:1000; Abcam, UK) against cleaved caspase-3 (catalog no. #ab32043), cleaved caspase-3 (ab2324), PARP1 (ab32064), SIRT1 (ab189494) and β-actin (ab7817). Next, membranes were incubated at room temperature for 1.5 h with horseradish peroxidase-conjugated goat anti-rabbit antibody (diluted 1:3000; Abcam). Finally, images were photographed using the Tanon ECL detection system (Bio-Tanon, Shanghai, China) and quantified using Image J software [18 (link)].

Western blotting was performed on the total protein extracts of infected Caco-2 cells pretreated with FBA. For the total protein fraction, the harvested cells were washed in cold phosphate-buffered saline (PBS) and lysed in protein lysis buffer (RIPA). Protein concentrations in cell extracts were determined using the Bradford assay (BioRad, Milan, Italy). Thirty micrograms of total lysates were loaded onto 10% SDS-PAGE and then transferred to nitrocellulose membranes (ImmobilonR-Transfer Membrane, Tullagreen, Carrigtwohill, Co). The membranes were blocked with 5% non-fat milk in PBS, pH 7.6, 0.2% Tween 20 (PanReac AppliChem) and probed overnight at 4 °C with the specific primary antibodies for Nf-KB p65 (1:1000; Cell Signaling, MA, USA) and Nfr2 (1:1000, #89443, AbCam, Cambridge, UK). After washing in PBS, pH 7.6 and 0.2% Tween 20, the membranes were incubated with a horseradish peroxidase-conjugated goat anti-rabbit antibody (1:2000; Abcam). The immunoblots were visualized using ECL detection kits, with enhanced chemiluminescence (Pierce, Rockford, IL, USA). A mouse β-actin antibody (1:5000; Elabscience) was used as the control for equal loading of total lysates.