Western Blot Analysis of Protein Expression

Cells were collected following the trypsin digestion, washed with PBS, and lysed with radioimmunoprecipitation assay (RIPA) lysis buffer with phenylmethane sulfonyl fluoride (PMSF). Total protein levels were determined by using BCA Protein Assay Kit (Pierce). The same amount of protein was taken from each sample, the same volume of 2× loading buffer was added, and the sample was cooked in boiling water for 10 min. Proteins were separated on 10% SDS-PAGE, and transferred to polyvinylidene fluoride (PVDF) membranes (Amersham), which were blocked with Tris-buffered saline with Tween 20 (TBST) solution containing 5% skimmed milk for 1 h, and the membranes were incubated with primary antibodies overnight. PVDF membranes were incubated with the secondary antibodies for 2 h, and the results were visualized using the ECL Plus Western Blotting kit (Amersham).
In the signaling pathway analyses, proteins were analyzed by using an automated western blot system (http://www.proteinsimple.com/simon.html). The following automatic ran was used: separate/immobilize/incubate with primary antibody/wash/ incubate with secondary antibody/wash/ incubate with enzyme substrate/expose [23 (link)–25 (link)].