Bio spectrum gel imaging system

Total protein samples were extracted from kidney tissues and NRK-52E cells using appropriate cold lysis buffer containing 1 mM PMSF following the manufacturer’s instructions. After determination of the contents, the proteins were separated by SDS-PAGE (8%–12%), transferred to PVDF membranes (Millipore, MA, USA), then blocked with 5% dried skim milk (Boster Biological Technology, Wuhan, China) and incubated overnight at 4 °C with the primary antibodies listed in Table 2. The membranes were incubated with an HRP-conjugated secondary antibody for 2 h at room temperature. And protein detection was performed based on an enhanced chemilumines-cence (ECL) method and photographed by using a Bio-Spectrum Gel Imaging System (UVP, Upland, CA, USA). Intensity values of the relative protein levels were normalized to GAPDH.

Total protein samples were obtained from the livers using the Tissue Protein Extraction Kit on the basis of the manufacturer’s instructions, and the protein content was determined using a BCA Protein Assay Kit. The protein samples (7 mg/mL) were denatured by mixing with an equal volume of 2× sample loading buffer and boiling at 100 °C for 5 min. The proteins were subjected to 10% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and transferred onto PVDF (polyvinylidene difluoride) membranes (Millipore, Burlington, MA, USA). After blocking the non-specific binding sites for 3 h with 5% dried skim milk in TTBS(10 mM TBS plus 1.0% Tween 20) at room temperature, the membranes were individually incubated overnight at 4 °C with the primary antibodies (Table 2). The membranes were then incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody for 2 h at room temperature at a 1:2000 dilution. Protein expression was detected by using an enhanced chemiluminescence (ECL) method and a Bio- Spectrum Gel Imaging System (UVP, Upland, CA, USA). The intensity values of the protein levels were normalized to GAPDH expression level.

Cells were lysed in RIPA lysis buffer, and the protein concentrations were determined using the BCA method. The samples were subjected to SDS‐PAGE and transferred to PVDF membranes (Millipore). After blocking in 5% skim milk, the membranes were probed with primary antibodies (Table 4) overnight at 4°C, followed by anti‐rabbit secondary antibody (Table 4) for 2 hours at room temperature. The protein bands were visualized using enhanced chemiluminescence (ECL) (Beyotime) on a BioSpectrum Gel Imaging System (UVP). Relative protein expression was normalized to GAPDH.

The HSC-T6 cells (2 × 10⁵ cells/mL) were plated in six-well plates and treated with dioscin (1.25, 2.5, and 5.0 μg/mL). Total proteins from different groups were extracted by cell lysis buffer containing PMSF. Next, the lysates were centrifuged at 12,000 × g for 10 min at 4°C. Then, the total proteins were loaded onto the SDS-PAGE gel (10–15%), separated electrophoretically and transferred to the PVDF membrane (Millipore, United States). The PVDF membranes were put into 5% dried skim milk for 3 h at room temperature, incubated overnight at 4°C with the primary antibodies (Table 2), then incubated with horseradish peroxidase-conjugated antibodies for 2 h at room temperature. Proteins were detected by enhanced chemiluminescence method and photographed using the Bio-Spectrum Gel Imaging System (UVP, United States), which were normalized with GAPDH as an international control. Three blots of each protein were performed, and five lanes were quantified. Where applicable, the image intensities of specific bands were quantified by Image-Pro Plus software (Media Cybernetics, United States). IOD value of target protein versus IOD value of GAPDH was used to eliminate the variation of protein expression.