Western Blot Analysis of Protein Expression

Cell lysates were separated in a 10% or 15% polyacrylamide gel and transferred onto a PVDF membrane (Millipore Corporation, Milford, MA, USA) [19] (link). The blot was subsequently incubated with 5% non-fat milk in PBS for 1 h to block non-specific binding, and probed with a corresponding antibody against a specific protein for 37°C at 2 h or overnight at 4°C, and then with an appropriate peroxidase conjugated secondary antibody for 1 h. After the final washing, signal was developed by ECL detection system and relative photographic density was quantitated by a gel documentation and analysis (AlphaImager 2000, Alpha Innotech Corporation, San Lean 189 dro, CA, USA) [20] (link).