CypA depletion attenuates ERK1/2 activity in M139 cells in vitro. ERK1/2 phosphorylation in CypA knockdown cells was determined by Western blot analysis. (A) Transient knockdown CypA decreased pERK1/2:ERK1/2 ratio in a time dependent fashion, and CypA protein level is positively correlated with ERK1/2 activity (phosphorylation). M139 cells (2 × 105 cells) were seeded in 6-well plate for 24 hours before being transfected with 100 pmole/mL siRNA against CypA or negative control siRNA for 6 hours before replacing with complete medium. Lipofectamine™ 2000 was used as a transfection reagent. Total proteins were collected at 24, 48, and 72 hours after transfection. (B) Stable knockdown CypA cells had lower ERK activity (phosphorylation) than the control cells. Protein samples were collected from M139-shV and M139-shCypA cells grown in complete medium for 24 hours. All samples were subjected to Western blot analysis for CypA, ERK1/2 and pERK1/2 detection, and b-actin was used as a loading control. Intensity of the immunoreactive bands was measured by ImageQuant TL software (GE Healthcare). Protein level of each sample was normalized to b-actin prior to comparison between samples. The pERK1/2:ERK1/2 ratio value reflects ERK1/2 activity.