Primary rabbit anti human antibodies

Cells were washed three times with phosphate-buffered saline (PBS). Proteins were extracted from the cells and quantified using a BCA Protein Assay Kit (Beyotime). Extracted proteins were loaded on 10% sodium dodecyl sulfate polyacrylamide gels, transferred to polyvinylidene fluoride membranes (Bio-Rad), and blocked with 5% nonfat milk powder. Membranes were incubated overnight with the following primary rabbit anti-human antibodies (Cell Signaling Technology, Beverly, MA, USA): anti-Nrf2 (1 : 2000), anti-HO-1 (1 : 2000), anti-NRF1 (1 : 2000), anti-PGC1α (1 : 2000), and anti-β-actin (1 : 5000). Membranes were incubated with goat anti-rabbit secondary antibodies. Protein signals were visualized using the ECL Western Blotting Detection System (GE Healthcare, Piscataway, NJ, USA). The gray values of the bands were analyzed using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).

3D Caco-2 cysts were lysed with RIPA buffer containing 1% protease inhibitor and 1% phosphatase inhibitor cocktails (Sigma-Aldrich, Amsterdam, NL). Protein concentration was equalized then subjected to 12% SDS-PAGE separation, and then transferred to PVDF membrane (GE healthcare, Amsterdam, NL). After blocking for 1 hour with 5% milk at RT, the membranes were then incubated overnight at 4 °C with primary rabbit anti-human antibodies (Cell Signalling Technology, Leiden, NL) against p-p38, p38, p-ERK1/2, ERK1/2, p-JNK and JNK with 1:1000 dilution, and then against horseradish peroxidase-conjugated goat anti-rabbit IgG (Cell Signalling Technology, Leiden, NL) with 1:4000 dilution. After final washes, membranes were incubated in ECL western blotting detection reagent, and detection was performed using Gel Doc XR + Imaging system (Bio-Rad).