Bca protein kit

Total protein was extracted from the tissue samples and cells using RIPA and phenylmethyl sulfonyl fluoride (PMSF). Protein concentrations were quantified using a BCA protein kit (Yeasen, Shanghai, China). Equivalent proteins were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked using 5% skim milk-TBST and incubated with primary antibodies at 4 °C overnight. The membranes were washed for 30 minutes, 10 minutes at a time in TBST, and incubated with secondary antibodies at room temperature for 2 hours. ECL chemiluminescence is conducted to detect protein signals. GAPDH was chosen as the internal reference protein. Antibodies against the following proteins were used: GAPDH (5174, CST), TOP2A (12286, CST and A4389, ABclonal), CHK1 (ab40866, Abcam), p-CHK1 (ab278717, Abcam), CDK1 (ab133327, Abcam), Cyclin B1 (ab32053, Abcam), E-Cadherin (3195, CST), N-Cadherin (13116, CST), Vimentin (5741, CST) and Slug (9585, CST).

Technician isolated total protein from transduced cells utilizing RIPA Lysis Buffer (RIPA Lysis Buffer Strong, Beyotime, China) through protease inhibitor (Phenylmethanesulfonyl fluoride, Beyotime, China). Technician gained protein concentration through BCA protein kit (Yeasen, Shanghai, China). We separated protein samples (15 μg) using electrophoresis with 10% SDS-PAGE gel to transfer them to PVDF membrane. Next, we blocked membranes with skim milk (5%) for 1 h. Technician incubated primary antibodies on blots at 4 °C overnight on a shaker. Primary antibodies used were: anti-BAX (1:5000, Proteintech, Wuhan, China), anti-BCL-2 (1:5000, Proteintech, Wuhan, China), anti-N-cadherin (1:5000, Proteintech, Wuhan, China), anti-PDK3 (1:1000, Affinity Biosciences, Jiangsu, China), anti-E-cadherin (1:5000, Proteintech, Wuhan, China) and anti-β-actin (1:10,000, Proteintech, Wuhan, China). β-actin was applied as internal reference. On the second day, we incubated blots through peroxidase-labeled secondary antibodies on shaker for one hour. Secondary antibodies used were: goat anti-mouse IgG (1:5000, Biosharp, Anhui, China) and goat anti-rabbit IgG (1:5000, Biodragon, Beijing, China). Then ECL Western Blotting Kit (Vazyme, Nanjing, China) was employed to visualize the protein bands. Lastly, we analyzed bands utilizing Image J software.