Bicinchoninic acid assay kit

First, cells were lysed in ice-cold lysis buffer supplemented with protease inhibitor cocktail and protein phosphatase inhibitor to extract total proteins. Protein concentration was determined using a bicinchoninic acid assay kit (Applygen, Beijing, China), and the protein was boiled in 1 × SDS loading buffer. Lysates were separated by 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% non-fat dry milk for 1 h and then incubated overnight at 4 °C with primary antibodies against phospho-AMPK (p-AMPK) (Thr172) (Affinity Biosciences, 1:500), AMPK (ABclonal, 1:500), or β-actin (Sigma, 1:10,000). After three washes for 10 min, the membranes were incubated with a horseradish peroxidase-linked secondary antibody (Cell Signaling Technology) for 1 h. The signal was visualized with ECL (Millipore).

Western blotting was performed according to a previously described protocol (21 (link)). Following 24 h treated with TBOPP or transfected with siRNA, the cells were collected, and total protein was extracted using RIPA lysis buffer (Beyotime Institute of Biotechnology). Total protein was quantified using a bicinchoninic acid assay kit (Applygen Technologies, Inc.). Equal amounts of proteins (40 µg per lane) were separated via 10% SDS-PAGE and transferred to a PVDF membrane. Subsequently, the membrane was blocked with 5% non-fat milk in TBS and 0.1% Tween-20 buffer for 1 h at room temperature. The membrane was incubated overnight at 4°C with primary antibodies targeted against: DOCK1 (dilution 1:1,000; cat. no. ab97325; Abcam) and GAPDH (1:1,000; cat. no. ab9485; Abcam). Following primary incubation, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies (dilution 1:500; cat. no. 7074; Cell Signaling Technology) at room temperature for 2 h. Protein bands were visualized using a Pierce™ ECL kit according to the manufacturer's protocol (Thermo Fisher Scientific, Inc.). Protein expression was semi-quantified using ImageJ software (version 1.8.0; National Institutes of Health). GAPDH was used as the loading control.