Bca protein kit

Proteins from colon and liver samples of all groups were extracted and quantified using RIPA Lysis Buffer (Beyotime, Shanghai) and BCA protein kit (Beyotime, Shanghai), respectively. Subsequently, 10 μL of proteins at the same concentration (5 mg/mL) were electrophoresed on 5–20% SDS-PAGE gels and transferred to the PVDF membranes (0.22 μm), which were then incubated with primary antibodies against occludin (1:1,000), claudin-1 (1:1,000), β-actin (1:1,000), KEAP1 (1:1,000), NRF2 (1:1,000), HO-1 (1:1,000) and NQO1 (1:1,000) (ABclone, China) for 16 h. Subsequently, membranes were washed with TBST for 30 min, followed by incubated with the secondary antibody (ABclone, China) for 2 h. After further washing with TBST for 30 min, protein bands were visualised using an ECL reagent (Beyotime, Shanghai). Finally, the protein bands of the image were analyzed by ImageJ software.

HCC cells were digested and isolated using trypsin (T4799, Sigma-Aldrich, St. Louis, MO, USA), and the total proteins were extracted by centrifugation after cell lysis in RIPA buffer (20101ES60, Yeasen, Shanghai, China) on ice. The protein concentration was determined using the BCA protein kit (P0012, Beyotime). Protein samples (20 µg/well) were loaded onto a 10% SDS-PAGE gel and electrophoresed before being transferred to a PVDF membrane. After transfer, the membrane was blocked with 5% bovine serum albumin at room temperature for 1 h. Subsequently, the membrane was incubated overnight at 4 ^∘C with rabbit antibodies: anti-MMP-2 (ab92536, 1:1000, Abcam, Waltham, MA, USA), MMP-9 (ab76003, 1:1000), Cleaved-caspase 3 (ab2302, 1:50), Bax (ab32503, 1:1000), Bcl-2 (ab182858, 1:2000), Ki67 (ab16667, 1:1000), and GAPDH (ab9485, 1:2500). The membrane was washed with TBST and then incubated with Goat Anti-Rabbit IgG (ab6721, 1:2000) at room temperature for 1 h. Following washing with TBST, the protein bands were developed and exposed using ECL (P0018S, Beyotime) chemiluminescent solution, and the gray value of each protein band was quantified using Image J 1.8.0 software, with GAPDH serving as an internal reference protein for relative quantitative analysis.