Bca protein detection kit

V. dahliae was inoculated into 4-week-old Arabidopsis thaliana plants by the method of Zhang et al. [24 (link)]. Then the leaves of the transgenic plant were taken at 6, 12, 24, 36, and 48 h, respectively. The activity of H₂O₂ and SOD was determined according to the kit’s instructions (Jiancheng Biotech Inc., Nanjing, China). The protein content was determined using the BCA protein detection kit (Sangon Biotech, Shanghai, China).

The refrigerated livers were washed with pre-cold PBS twice and centrifugation at 3000× g for 5 min at 4 °C, then removed the supernatant. Total protein extracts were obtained by homogenizing liver in RIPA lysis buffer (Sigma Aldrich) supplemented with protease inhibitor cocktail and phosphatase inhibitors. After centrifugation, the supernatant was collected and stored at − 80 °C. Protein concentration was determined by the BCA protein detection kit (Sangon Biotech, Shanghai, China). Western blot analysis was performed as previously described by Han et al. The primary antibodies were used: caspase-3 (ZenBio, Chengdu, China), caspase-9 (ZenBio), β-actin (Abcam, Cambridge, MA, USA), Bax (ZenBio), Bcl-2 (Santa Cruz, Heidelberg, Germany), LC3B (Sigma), P62 (Santa Cruz), beclin-1 (Sigma), PI3K/Akt/mTOR/70S6K protein and phosphorylated antibody were purchased from Bioss Biotechnology Co. Ltd. (Bioss, Beijing, China). The secondary antibodies used were as follows: mouse anti-rabbit (Sigma), goat anti-rabbit (Sigma), mouse anti-rabbit horseradish peroxidase (HRP) (Zenbio). The enhanced chemiluminescence (ECL) kit (Beyotime, Jiangsu, China) was used to capture the bands via a CanoScan LiDE 100 scanner (Canon, Tokyo, Japan), and western blots were analyzed by Image J software (Bethesda, MD, USA, 2007).

Protein was extracted by using RIPA buffer (Sango Biotech, Shanghai, China), measured using a BCA protein detection kit (Sango Biotech, Shanghai, China), separated on a 10% SDS-polyacrylamide gel (PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA). Then, the membrane was blocked in 5% milk dissolved in 1× TBST at room temperature (RT) for 1 h and incubated with primary antibodies at 4 °C overnight. On the following day, the membrane was incubated in secondary antibody at RT for 1 h, and the results were visualized using an enhanced chemiluminescence detection kit (CWBIO, Beijing, China).