Ecl luminescent solution

Determination of tight junction proteins was referred to the method of Gao et al. [27 (link)]. HT–29 cells were inoculated with 4 × 10⁵ cells/well in 6–well plates for 48 h. Three yeast species (re–suspended with RPMI–1640 medium without fetal bovine serum and antibiotics), including S. cerevisiae I4, C. lusitaniae 30 and P. kudriavzevii 11 adjusted to 1 × 10⁷ CFU/mL were added and incubated for 2 h, then the supernatant was discarded and washed twice with PBS. Under the condition of ice bath, the bottom protein was scraped by a cell scraper and its concentration was determined by bicinchoninic acid (BCA) kit after pretreatment. After separated by SDS gel electrophoresis, the protein was transferred to polyvinylidene fluoride (PVDF) membrane by wet method. Then the membranes were incubated with Occludin, Claudin–1, ZO–1 and β–actin antibody (diluted as 1:1500) at 4 °C overnight and further incubated with corresponding secondary antibody at a dilution of 1:2000. At last the protein bands were visualized by Meilunbio ECL Luminescent Solution. β-actin was used to be the internal control protein.

Total protein samples were prepared using RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors and quantified by the BCA protein assay kit. Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight region of the target protein indicated by the pre-stained Marker was cut off and transferred to PVDF membrane or NC membrane, and the skimmed milk powder was closed at room temperature for 2 hours, and the primary antibody was added at the end of closure and incubated in the refrigerator at 4°C overnight. The primary antibodies are shown in Table 4. Then incubation with secondary antibody IgG (1:10,000) and preparation of ECL luminescent solution (meilunbio, Dalian, China) was performed for strip exposure. The strips were exposed using a 5,200 Tanon imaging system (Tanon science and technology, Shanghai, China). The developed protein bands were further quantified by ImageJ software and then compared with the grayscale values of the internal reference gene β-tubulin to calculate the relative expression levels of the target proteins measured in each of the above experimental groups.

Total protein was extracted by RIPA Lysis Buffer (Sangon Biotech, Shanghai, China) and quantified with BCA Protein Assay Kit (Sangon Biotech). The same amount of protein was isolated by 10% SDS-PAGE gel and transferred to PVDF membrane (Beyotime, Shanghai, China). Membranes were immersed in 5% nonfat milk and then incubated with primary antibodies, including CXCL5 (ab9802, 1:10,000), E-cadherin (ab40772, 1:40,000), N-cadherin (ab18203, 1:1000) and GAPDH (ab9485, 1:2500). After incubating with Goat Anti-Rabbit IgG H&L (HRP) (ab205718, 1:50,000), the protein signals on the membranes were analyzed using ECL luminescent solution (Meilunbio, Dalian, China). All antibodies were bought from Abcam (Cambridge, MA, USA).

After inoculation, the cells were cultured in complete medium at a density of approximately 70%, and the corresponding treatments were carried out according to the groups. Western blotting was carried out according to previous research protocols, and RIPA (Solarbio, CN) was used to extract the total proteins. A BCA protein assay kit (Beyotime, CN) was used to determine the protein concentration, and the same amount of protein (30 μg) was loaded into 10% SDS–PAGE gels for electrophoresis under constant pressure. The protein was then transferred to the PVDF membrane under constant current, sealed with 8% skimmed milk powder for 1.5 hours, incubated with the primary antibody (β-actin, AMPK, p-AMPK, BIP, and cleaved-caspase3, 1 : 1000 dilutions; CST, USA) for 12–16 hours, washed on a shaker with TBST 4 times for 10 minutes each time, and then washed with the residual secondary antibody on a shaker again with the corresponding secondary antibody (1 : 10000 dilutions, KPL, USA). The generated protein bands were exposed and visualized on a Fusion Fx machine in a dark room using ECL luminescent solution (Meilunbio, CN).