FIG 7.

GA- and stress-induced degradation of MLK3 requires CHIP. (A) SKOV3 cells were transfected with nonspecific or CHIP siRNA oligonucleotides and then treated with GA (10 μM) for 3 and 6 h. Whole-cell extracts were immunoblotted with anti-MLK3, anti-CHIP, and anti-β-actin antibodies. The MLK3 band intensity for each time point was normalized to the band intensity for β-actin and expressed as a percentage of the value for untreated cells. (B) HEK293 cells were transfected with CHIP and nonspecific siRNA oligonucleotides as described above for panel A and then subjected to a 42°C heat shock for 30 or 60 min. The cell lysates were immunoblotted with the indicated antibodies. The MLK3 band intensity for each time point was analyzed as described above for panel A. (C) HEK293 cells were transfected with CHIP and nonspecific siRNA oligonucleotides as described above for panel A and then treated with 0.25 M sorbitol for 2 or 4 h. The cell lysates were immunoblotted with the indicated antibodies. The MLK3 band intensity for each time point was analyzed as described above for panel A.