Quantitative Western Blot Analysis

Western blot analysis was carried out as described earlier [13 (link)]. Briefly, 4T1-Qt cells were lysed using RIPA buffer, and the resulting lysate was precipitated (15 min, 14,000× g). Sample buffer 5× was added to different amounts of cell lysate, heated at 95 °C, then cooled on ice. Samples were loaded onto a gel and electrophoresed (80 V for 25 min and 100 V for 1.5 h); then, the gel was transferred into a transfer buffer. The nitrocellulose membrane was activated and placed over the gel. The transfer was carried out in a chamber filled with transfer buffer for 1 h at 100 V. Then, the membrane was washed three times to remove transfer buffer residues in PBST. To prevent nonspecific binding, the membrane was incubated in a PBST solution with 5% non-fat milk for 2 h and washed again. The membrane was incubated with anti-DYKDDDDK Tag antibodies (1:1000, BioLegend, San Diego, CA, USA) for 2 h, followed by washing three times. After that, alkaline horseradish peroxidase conjugated secondary antibodies (1:1000, goat anti-mouse IgG, Santa Cruz Biotechnology,) were added. Clarity Max Western ECL Substrate kit (BioRad, Hercules, CA, USA) was used to reveal the result. The results were registered with the ChemidocMP Imaging system (BioRad, Hercules, CA, USA).