Image station 4000 mm pro system

Cells were washed and then added into cell lysis buffer (Beyotime Biotechnol, Shanghai, China) with 1 mM diethyl pyrocarbonate (PMSF). Sample proteins were extracted with lab-made protein lysate and heated at 80°C for 5 minutes, followed by 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). When electrophoresis was complete, proteins in the 12% gel were blotted onto a polyvinyl difluoride (PVDF) membrane and then sealed with 0.05% TBS Tween supplemented with 3% FCS for at least 1 h, followed by incubation of the PVDF membrane with a specific primary antibody overnight at 4°C. Eight to twelve hours later, the PVDF membrane was washed three times, 10 minutes per wash, and then incubated with an HRP-conjugated secondary antibody at room temperature for 1 hour. An image was obtained using chemiluminescence substrate (Pierce, IL, USA) and an Image Station 4000 mm PRO system (Kodak).

Cell pellets were lysed in cell lysis buffer (Beyotime Biotechnol, Shanghai, China) containing 1 mM phenylmethyl-sulfonylfluoride (PMSF) and samples were boiled for 5 minutes. About 25 μg of protein was separated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto a polyvinyl difluoride (PVDF) membrane. After blotting, the membrane was blocked with 5% nonfat dry milk in Tris-Buffered Saline containing Tween 20 (TBST) for 2 hours and incubated overnight at 4°C with primary antibody. The membrane were then washed in TBST and incubated for 1 hour with the HRP-conjugated secondary antibodies (CST). Imaging of the blot was performed with super signal west pico chemiluminescence substrate (Pierce, IL, USA) using Image Station 4000 mm PRO System (Kodak). Protein band intensities were measured by Image Station 4000 mm PRO software. The control group was set as 100 to allow comparisons.

Total cell lysates were prepared and analysed by western blot as previously described20 (link). The specific primary antibodies were used to detect the markers at 4 °C overnight. The blots were visualised using ECL (Promega, Madison, WI, USA) on a Kodak Image Station 4000 mm Pro System (Kodak, Rochester, NY, USA). The density of the bands was quantified by densitometric analysis using the Image Tool (version 3.0) system.