Bca assay kit

Tissues and cells were lysed in RIPA buffer supplemented with protease inhibitor and phosphatase inhibitor (Thermo Scientific). The protein concentration was evaluated by using BCA Assay Kit (Biyuntian) and then was denatured by using loading buffer (5×) at 100°C for 5 minutes. Proteins with equal amounts (30 μg) were separated by SDS‐PAGE. Primary antibodies used were listed as follows: anti‐E‐cadherin antibody (1:1000, #3195; Cell Signaling Technology); anti‐vimentin antibody (1:1000, #5741; Cell Signaling Technology); anti‐ZEB1 antibody (1:1000, #3396; Cell Signaling Technology); and anti‐SIX‐1 antibody (1:1000, #16960; Cell Signaling Technology). Then, the PVDF membranes were incubated with antimouse or anti‐rabbit HRP‐conjugated secondary antibody (1:1000; ZSGB‐bio). An anti‐β‐actin antibody (1:5000, A5441; Sigma‐Aldrich) was used as an internal control.

Protein CrlTA complexes with a hexahistidine tag at the N-terminus of the CrlTA complex without any tag were purified using E. coli BL21 with pET28b-His-crlTA and pET28b-crlTA. Strains were grown in LB with kanamycin (50 μg/ml) and were induced with 1 mM IPTG at OD₆₀₀ ~ 0.1 for 5 h. Cells were collected and resuspended in lysis buffer [50 mM potassium phosphate buffer (pH 8.0), 300 mM NaCl, and protease inhibitor cocktail (Sigma-Aldrich, United States)]. Samples were sonicated using a Sonic Dismembrator (Ningbo Dongzhi, China) at level 2 for 5 min on ice. Ni-NTA resin (Qiagen) was used according to the manufacturer’s protocol. Purified proteins were desalted by passage on disposable Sephadex G-25 prepacked PD-10 columns pre-equilibrated in 20 mM Tris–HCl buffer (pH 8.0), and the protein concentration was measured by the Bi Yuntian BCA assay kit (Haimen, Jiangsu, China). Tricine–SDS-PAGE was performed as previously described (Baba et al., 2006 (link)). A total of 20 μg of protein from each sample was loaded for Tricine–SDS-PAGE.

Mice were sacrificed by cervical dislocation, and their hippocampal and PFC tissues were immediately dissected from the left and right brains at 4°C and placed in PBS buffer (0.01 M). The hippocampal and PFC tissues were separately placed into EP tubes and stored at −80°C until use in the biomarker assays. The hippocampal and PFC samples were lysed and homogenized using tissue lysis buffer (containing protease and phosphatase inhibitors) in an ice bath for at least 30 min. Next, the total homogenate was centrifuged at 13,000 rpm for 15 min at 4°C. The supernatants were collected, divided into new tubes, and stored at −80°C for use in Western blot and oxidation-associated biomarker assays.
The total protein concentration in each sample was quantified using a bicinchoninic acid (BCA) assay kit (Shanghai Biyuntian Biotechnology Co. Ltd.) based on a standard curve. The MDA, GSH-PX, and T-AOC contents were determined using MDA, GSH-PX, and T-AOC kits, respectively, according to the manufacturer's recommendations with a slight modification in dosage. The MDA content was expressed as nmol per milligram of protein (nmol/mg), and the GSH-PX and T-AOC activities were expressed as units per milligram of protein. Apart from these methods, the other protocols utilized were basically similar to those described above for serum detection.