Quantitative Western Blotting Analysis

Western blotting was performed as previously described [17 (link)]. Primary antibodies included p-AKT Ser473, AKT, p-ERK Thr204/205, ERK, p-MEK Ser217/221, MEK, cyclin D1, cyclin D3, p-GSK3β, GSK3β, BIM and GAPDH (all from Cell Signaling Technology, Danvers, MA). The immunoreactivity was revealed by use of an ECL2 kit (Pierce Rockford, IL) and scanning of the blots by the Typhoon scanner (Amersham Biosciences Co, Piscataway, NJ). The intensity of the p-MEK bands were determined by the ImageQuant software and were normalized between the two blots by considering the reference sample (one of the samples of the first blot duplicated on the 2nd blot). The relative level of p-MEK for each sample was determined by the ratio of each sample over the average intensity of all the samples.