Bca protein quantitation kit

Western blot was performed as described previously [17 (link)]. RIPA buffer (R0278, Sigma–Aldrich, USA) was applied to lyse and extract the total protein from cells. After the protein concentration was measured by BCA protein quantitation kit (55R-1544, Fitzgerald, USA), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels were exploited to separate the protein (45 μg) and marker (5 μL; G2086, Servicebio, China), which was then moved to polyvinylidene fluoride membranes (24937, Sigma-Aldrich, China). The membranes were sealed by defatted milk and subsequently incubated with primary antibodies (Table 2) at 4°C overnight. Next, the membranes were incubated with the goat anti-rabbit (ab97051, 1 : 5000, Abcam) or rabbit-anti-mouse secondary antibody (ab6709, 1 : 2000, Abcam) for 2 h. An excellent chemiluminescent substrate detection kit (E-BC-R347, Elabscience, China) was used to measure the protein bands, and an eZwest Lite auto western blotting system (Genscript, Piscataway, NJ, USA) was employed to scan the bands.

Total protein in MPC-5 cells was extracted using the RIPA reagent (89901, Thermo Scientific, USA). Then, the concentration of protein samples was quantified by BCA protein quantitation kit (55 R-1544, Fitzgerald, USA). Next, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels were exploited to separate the protein sample, which was then moved to polyvinylidene fluoride membranes (24937, Sigma-Aldrich, China). The membranes were sequentially soaked in blocking solution to reduce nonspecific binding and incubated with primary antibodies against Cleaved (Clea)-caspase-3 (#9661, 1:1000, 17, 19 kDa, Cell Signaling Technology, USA), AKT1 (ab179463, 1:10,000, 56 kDa, abcam, UK), phosphorylated (p)-AKT1 (ab81283, 1:10,000, 56 kDa, abcam, UK), and GAPDH (ab9485, 1:2500, 37 kDa, abcam, UK) at 4 °C overnight. Next, the membranes were incubated with the secondary antibodies including goat anti-rabbit IgG (ab97051, 1:5000, abcam) and rabbit anti-mouse IgG (ab6709, 1:2000, abcam) for 2 h. Excellent Chemiluminescent Substrate Detection Kit (E-BC-R347, Elabscience, China) was used to measure the protein bands, and ImageQuant LAS 4000MINI Ultra-Sensitive Chemiluminescence Imager (Sinopharm Chemical Reagent Co. Ltd., China) was employed to scan the bands. GAPDH served as the loading control of this assay.