Ab7063

Extraction of total protein was conducted utilizing QIAzol™ lysis reagent (Qiagen, CA). 40 μg protein was subjected to SDS–PAGE as well as transferred onto PVDF membranes (Millipore, MA). Thereafter, the blots were blocked with 5% blotting grade milk lasting 1 h and were incubated with a primary antibody against ERCC6 (#24291-1-AP; 1:500; Proteintech, Wuhan, China), GAPDH (#10494-1-AP; 1:10000; Proteintech), Bax (#60267-1-Ig; 1:5000; Proteintech), cleaved caspase-3 (#19677-1-AP; 1:500; Proteintech), Bcl-2 (#26593-1-AP; 1:1000; Proteintech), p21 (#10355-1-AP; 1:1000; Proteintech), p27 (#25614-1-AP; 1:1000; Proteintech), 4-HNE (#ab46545; 1:3000; Abcam, United States), and Nrf2 (#16396-1-AP; 1:500; Proteintech) or Keap1 (#10503-2-AP; 1:2000; Proteintech) overnight at 4°C. The following day, the blots were incubated in horseradish peroxidase-conjugated goat anti-rabbit (#ab7090; 1:5000; Abcam) or anti-mouse secondary antibody (#ab7063; 1:5000; Abcam) lasting 1 h at room temperature. Thereafter, the blots were developed with enhanced chemiluminescence. The gray value was quantified via ImageJ software.

The cells and cecum tissues of the above groups and mice were ground and homogenized and added with lysate, and lysed with ultrasound. After that, the samples were centrifuged for 10 min. A small amount of sample was taken to determine the protein concentration using bicinchoninic acid (BCA) protein quantification kit (Thermo Fisher Scientific Inc., Waltham, MA, United States) on the microplate reader. For each sample, 50 μg protein was loaded onto a 10–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel with tris-glycine running buffer, and transferred to polyvinylidene fluoride (PVDF) membranes, which was blocked with 5% (m/v) skimm milk or bovine serum albumin (BSA) for 2 h. The membranes were incubated with primary antibodies (Table 2, all from Abcam Inc., Cambridge, MA, United States) at 4°C overnight. The next day after washing with tris-buffered saline with Tween (TBST), the membranes were incubated with goat anti-mouse secondary antibody (1:2,000, ab7063, Abcam) for 2 h. After visualization using enhanced chemiluminescence, the membranes were imaged using the gel imaging system. The band intensity was quantified with Image-Pro Plus 6.0 software (Olympus Optical Co., Ltd, Tokyo, Japan).