Pierce bca protein assay kit

Forty-eight hours after transfection, DF-1 cells were lysed in RIPA buffer (Beyotime, Nantong, China) supplemented with 100 mM phenyl methane sulfonyl fluoride (PMSF) to exact total protein. Protein concentrations were measured by the Pierce BCA Protein Assay Kit (Transgen, Shanghai, China). An equal amount of protein was separated by 12% SDS-polyacrylamide gel electrophoresis (Beyotime, China) and blocked with 5% skim milk for 1h. Then, the membranes were separately probed with p-JUN (ABclonal, AP0048), p-FOS (ABclonal, AP0038), p-JNK1 (ABclonal, AP0631), Bcl-2 (ABclonal, A19693), Caspase8 (ABclonal, A0215), Caspase9 (ABclonal, A18676), Caspase3(ABclonal, A19654), GAPDH (Abmart, M20024) overnight at 4°C with a final dilution of 1:5000 (v/v). Finally, the membrane was incubated with the secondary antibody for 1h after TBST washing. The enhanced chemiluminescence (ECL) detection system (Bio-Rad) was used to detect protein expression.

Small intestinal tissues were homogenized in RIPA buffer (Solarbio, China) with a mixture of protease inhibitors. The Pierce BCA Protein Assay Kit (TransGen Biotech, China) was used to quantify protein concentration. Protein samples were subjected to electrophoresis using on 10% sodium dodecyl sulfate-polyacrylamide gels and transferred to PVDF membranes (MilliporeSigma, Burlington, MA, United States). Subsequently, the membranes were blocked with skimmed milk powder (Solarbio) and incubated overnight at 4°C with the following primary antibodies: anti-ZO-1 (ab96587, 1:1000 dilution; Abcam), anti-Occludin (A2601, 1:2000 dilution; ABclonal, Woburn, MA, United States), anti-Claudin-1 (ab307692, 1:1000 dilution; Abcam:1000), and anti-GAPDH (ab181602, 1:1000 dilution; Abcam). After five washes with TBST for 5 min each, the membranes were incubated with the respective secondary antibodies (goat anti-rabbit; SA00001-2, Proteintech, Rosemount, IL, United States) for 1 h at room temperature. Protein bands were visualized using a high-sensitivity ECL chemiluminescence kit (NCM Biotech, China) and gel imager (Bio-Rad Laboratories, Hercules, CA, USA), and band intensity was quantified using ImageJ software version 2.3.0 (National Institute of Health, Bethesda, MD, USA).