Ez western lumi femto western blot detection kit

The cells were washed with PBS and centrifuged at 1000× g for 5 min at 4 °C. The obtained pellets were resuspended in RIPA buffer with protease and proteasome inhibitors, incubated on ice for 20 min, sonicated, and centrifuged at 20,000× g for 20 min at 4 °C. The supernatants were separated using 8% or 10% SDS-PAGE and then transferred to nitrocellulose membranes. The membranes were incubated overnight at 4 °C with primary antibodies against MITF, GAPDH (Thermo Fisher Scientific, Rockford, IL, USA), tyrosinase, TRP1, TRP2, β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), cAMP-responsive element binding protein (CREB), or pCREB (Cell Signaling Technology, Danvers, MA, USA), which was followed by incubation with the appropriate secondary antibodies (Thermo Fisher Scientific, Rockford, IL, USA). The blots were developed by using the EZ-Western Lumi Femto™ western blot detection kit (Daeil Lab Service, Seoul, Korea).

Cells were washed with PBS and centrifuged at 1,000 × g for 5 min at 4°C. Pellets were resuspended in radioimmunoprecipitation assay (RIPA) buffer with protease and proteasome inhibitors, incubated on ice for 20 min, sonicated, and centrifuged at 20,000 × g for 20 min at 4°C. Cell lysates were subjected to SDS-PAGE, followed by transfer to a polyvinylidene difluoride or nitrocellulose membranes (Merck Millipore). Membranes were incubated overnight at 4°C with the specific primary antibodies, followed by incubation with secondary antibodies. Blots were developed using the EZ-Western Lumi Femto™ western blot detection kit (DAEILLAB Service).