FIG 1.

GA stimulates MLK3 ubiquitination and reduces the level of endogenous MLK3 protein. (A) SKOV3 cells were treated with GA (10 μM) for the indicated time periods, and whole-cell extracts were immunoblotted with the indicated 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) SKOV3 cells were treated as described above for panel A. MLK3 mRNA transcript levels were analyzed by quantitative RT-PCR, normalized to the β-actin transcript levels, and expressed as a percentage of the value for control, untreated cells. The values represent the means ± standard deviations (n = 3). No statistically significant differences in mRNA levels were observed between the GA-treated and untreated cells. A P value of <0.05 was considered statistically significant. (C) SKOV3 cells were treated with cycloheximide (CHX) (50 μM) alone or cycloheximide (50 μM) and GA (10 μM) for the indicated time periods. The MLK3 band intensity was determined for each time point as described above for panel A. (D, top) Endogenous MLK3 was immunoprecipitated (IP) from cell lysates with anti-K63 ubiquitin (K63-Ub) or anti-K48 ubiquitin (K48-Ub) antibodies, and the immunoprecipitates were immunoblotted (IB) with anti-MLK3 antibody. (Bottom) The cell lysates were also immunoblotted with anti-MLK3 and anti-β-actin antibodies. (E) HEK293 cells were treated with vehicle or MG132 (10 μM) for the indicated time periods. MLK3 and β-actin protein levels were assessed by immunoblot analysis with anti-MLK3 and anti-β-actin antibodies. (F) SKOV3 cells were untreated or treated with MG132 (10 μM) for 2 h and then treated with GA (10 μM) for 6 h. Immunoblotting of cell extracts was performed as described for panel E.