Western Blot Protein Detection Protocol

Whole cell lysates were prepared, and total proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA). The blots were incubated with 5% skim milk at room temperature for 1 h, then stained with primary antibodies, and then incubated with peroxidase-conjugated secondary antibody. The proteins of interest were detected by ECL Western blotting detection reagents (GE Healthcare, Piscataway, NJ, USA). The protein expression levels were analyzed using an Azure c400 system and AzureSpot Analysis Software (Azure Biosystems, Dublin, CA, USA) following the manufacturer’s instructions [55 (link)].