Western Blotting Analysis Protocol

Western blotting analyses were conducted followed routine protocol described previously (6 (link), 7 (link), 19 (link)–21 (link), 23 (link)). Consistent amount of protein samples were resolved on a 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE). A 20 μg quantity of protein was loaded in each well with 5x sample buffer, and the protein samples were resolved with electrophoresis at 100 V for 2 h. The resolved proteins were transferred from the polyacrylamide gel to Millipore Immobilon-PSQ Transfer PVDF membranes (Millipore, Billerica, MA, USA) with the Mini Trans-Blot® Cell (Bio-Rad Laboratories). The membranes were blocked with a solution of 2% FBS in Tris-buffered saline and incubated with primary antibodies to pERK1/2 (GeneTex, Inc., Hsinchu, Taiwan), Cyclin D1 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), ERK1/2 (Santa Cruz Biotechnology), and α-Tubulin (Novus Biologicals, Littleton, CO, USA) at 4°C overnight and washed; the proteins were detected with HRP-conjugated secondary antibodies and Immobilon™ Western HRP Substrate Luminol Reagent (Millipore). Images of the western blots were visualized and recorded with an Amersham Imager 600 (GE Healthcare, Chicago, IL, USA). The blots were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).