Figure 3.

MEKK2 inhibits heat- and calpain-mediated degradation of STK38. (A) COS-7 cells were transfected with human STK38-V5 alone, or with FLAG-MEKK1 or FLAG-MEKK2. Forty-eight hours after transfection, the cells were heated to 44 °C for 20 min or left untreated as controls and harvested. (B) COS-7 cells were transfected with human STK38-V5 alone, or with FLAG-MEKK2 (WT) or FLAG-MEKK2 (KM). Forty-eight hours after transfection, the cells were harvested. (C) HeLa cells were transfected with the scramble oligonucleotides control (scr) or MEKK2-specific siRNA. Forty-eight hours after transfection, the cells were heated to 44 °C for the indicated times or left untreated as controls and harvested. (D) HeLa cells were transfected with FLAG-MEKK2. Forty-eight hours after transfection, the cells were heated to 44 °C for the indicated times or left untreated as controls and harvested. The MEKK2 activity was measured by immune complex kinase assay with an anti-FLAG antibody using GST-MKK4 as the substrate. (E) GST-STK38 was incubated with calpain I (0.07 units of calpain I in each lane) in the absence or presence of GST-active MEKK2 for 15 min at 30 °C. Cell lysates or in vitro reaction products were analysed by western blotting with the antibodies against the indicated proteins. A representative image of western blot is shown (see Supplementary Fig S6 for corresponding full-length image). Relative levels of STK38 were determined from the western blot using Image J software. Data are presented as the mean ± standard deviation of three independent experiments. Statistical significance was determined by the Student’s t-test (**P < 0.05).