Electrochemiluminescence kit

After the extraction of total proteins from NSCLC cells with RIPA lysis buffer (Beyotime, Shanghai, China), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed, and then the protein was transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked by 5% defatted milk for 2 h at room temperature and then incubated with primary antibodies including anti-E-cadherin (1:1000; ab40772, Abcam, Shanghai, China), N-cadherin (1:1000; ab76057, Abcam), Snail (1:1000; ab216347, Abcam), Twist1 (1:1000; ab187008, Abcam), ZEB1 (1:1000; ab203829, Abcam), Vimentin (1:1000; ab92547, Abcam), DDX5 (1:1000; ab126730, Abcam) and GAPDH (1:1000; ab181602, Abcam) at 4°C for 12 h. Subsequently, the membranes were incubated with HRP-labeled secondary antibody (1:2000; Beyotime, Shanghai, China) at room temperature for 2 h. Electrochemiluminescence kit (Promega, Madison, WI, USA) was used to develop the protein bands.

The cell lysates of the chondrocytes were then loaded onto a 5%–20% gradient sodium dodecyl sulfate-polyacrylamide gel, subjected to electrophoresis under reducing conditions, and blotted onto a polyvinylidene difluoride membrane (Bio-Rad). The blots were blocked with a solution of 3% nonfat dry milk/2PBS/0.1% Tween-20 at room temperature, rinsed twice with PBS/0.1% Tween-20, and incubated with 1:200 diluted polyclonal anti-caspase 3 or anti-cleaved caspase 3, 8, and 9 antibodies (St. John's Lab, London, UK), followed by 1:5000 diluted anti-rabbit immunoglobulin G horseradish peroxidase (HRP) (Amersham GE, Taipei, Taiwan). Detection of actin by anti-actin antibodies (Santa Cruz, Dallas, TX, USA) was utilized as a loading control. Membranes were rinsed three times in PBS/0.1% Tween-20. Signals were detected with HRP using an electrochemiluminescence kit (Promega, Fitchburg, WI, USA). The intensities of cleaved caspase 3 were quantified using ImageJ processing [37 (link)].