Bca protein evaluation kit

Western blotting was used for determining the protein concentration in the tissues of the spinal cord. In brief, we lyse tissues from various groups with Radio-Immunoprecipitation assay lysis buffer (Beyotime Bio Inc, Shanghai, China) and measure protein quantities with the BCA protein evaluation kit (Beyotime Bio Inc). Later, using a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Beyotime Bio Inc), protein specimens of identical amounts were separated and moved to membranes fabricated from polyvinylidene difluoride (Beyotime Bio Inc). Following blocking with 5% non-fat dry milk, the membranes were treated at 4° C during the night hours with primary antibodies. GAPDH (Santa Cruz Bio Inc, USA), caspase-8 (Santa Cruz Bio Inc, USA) and STAT3 (Santa Cruz Bio Inc, USA) were the set of primary antibodies employed in the work. Then, to bind with the horseradish peroxidase, the appropriate secondary antibodies were added. The blotting findings were seen using an ECL detection reagent provided by 7 Sea Biotech, Shanghai, China. A Gel-Pro-Analyzer (Media Cybernetics, USA) was implemented to quantify the data.

The levels of C Foxg1, cyclin D1, PCNA, and GAPDH protein were detected by WB. GDF-5 was treatment EpSCs for 24 h, and then collect cells. EpSCs protein samples were prepared using RIPA lysis buffer (Beyotime, P0013B, China), which contained protease and phosphatase inhibitors. It was quantified using BCA protein evaluation kit (Beyotime, P0012S, China). Next, 30 μg/each sample of protein was loaded onto 10% SDS-PAGE and transferred to PVDF membrane (Beyotime, FFP24, China). Then, the membrane was blocked with a 5% milk solution (w/v) at room temperature for 2 h. Then, the primary antibody was incubated overnight at 4 °C. The primary antibody was diluted according to the following ratio: PCNA (ab92552, 1:5000), Foxg1 (ab196868, 1:5000), cyclin D1 (ab16663, 1:5000), and GAPDH (ab181606, 1:10,000). All antibodies were purchased from Abcam (Cambridge, Massachusetts, USA). After incubating the primary antibody, the membrane was washed three times and incubated with goat anti-rabbit IgG (H + L) (1:10,000) (Abcam, ab6702) for 1 h. The bands were visualized by using the BeyoECL Plus (Beyotime, P0018M, China), and the bands were detected using Image Quant LAS 4000 s (GE, USA) [33 (link)].

The treated cells were obtained and resuspended in cell lysate (Thermo) on ice for at least 30 min. The cell lysate was centrifuged at 12 000 × g for 10 min at 4°C. The supernatant was obtained, mixed with 5 × loading buffer, and boiled for 10 min. After boiling, the resulting supernatant containing total protein was quantified using the BCA protein evaluation kit (Beyotime P0012). An 8%, 10%, or 15% polyacrylamide gel (depending on the protein's molecular size to be analyzed) was used to separate a 50 µg total protein sample. The separated sample was transferred to a PVDF membrane (Millipore). The membrane was blocked using 5% skimmed milk and incubated with primary antibodies (1: 1000 in TBST) of cleaved caspase 3, cleaved caspase 8,Bax, Bcl‐2, P‐AMPK, P‐mTOR, p‐p70s6k, and GAPDH. Secondary antibodies coupled with horseradish peroxidase were then introduced into the membrane. The membrane was then incubated. Enhanced chemiluminescence (ECL) reagent (Pierce, Thermo Fisher Scientific) was used to detect protein expression.