Figure 2

D38A and D38K mutants impaired the cellular functions of PP2Cα.

(a) The cellular effects of PP2Cα-WT, D38A and D38K mutant overexpression on EGF-induced ERK phosphorylation in HEK293 cells. The HEK293 cells were cultured in DMEM containing 1% penicillin/streptomycin, 10% fetal bovine serum and 25 mM glucose at 37°C and 5% CO₂. The cells were transfected with the full-length wild-type PP2Cα and its mutants (D38A and D38K). After 36 hours of transfection and 12 hours of starvation, the HEK293 cells were stimulated with 5 ng/ml EGF for 5 min at 37°C. The levels of ERK1/2 phosphorylation were monitored using a phospho-ERK1/2-pT²⁰²pY²⁰⁴ antibody. Total ERK1/2 was used as a loading control. The western blot shows a representative result from at least three experiments. (b) Statistical analysis of Fig.2a. *: P < 0.05, cells transfected with PP2Cα-WT compared with control cells. #: P < 0.05, cells transfected with D38A or D38K compared with cells transfected with PP2Cα-WT. The data showed are the average of at least three independent experiments. (c) The cellular effects of PP2Cα-WT, D38A and D38K on sorbitol-induced JNK phosphorylation in U251 cells. The U251 cells were cultured in medium similar to the HEK293 cells. 36 hours after transfection, the U251 cells were starved for 12 hours and then stimulated with 0.4 M sorbitol for 30 min at 37°C. The phospho-JNK-pT²²¹pY²²³ levels were monitored by western blot. The data shows a representative result from at least three experiments. (d) Statistical graph of Fig.2c. *: P < 0.05, cells transfected with PP2Cα-WT compared with control cells. #: P < 0.05, cells transfected with D38A or D38K compared with cells transfected with PP2Cα-WT. The data showed are the average of three independent experiments at least.