FIGURE 1.

Effect of MLK3 on β-catenin expression in various human cell lines. A, subconfluent populations of HEK-293 cells were transiently transfected with HA-tagged β-catenin alone (second lane) or in combination with M2-tagged MLK3-WT (third lane) or M2-tagged MLK3-K/A (fourth lane). Equal amounts of total protein isolated from these cells were subjected to Western blot analyses with antibodies against HA (for ectopic β-catenin) or M2 (for ectopic MLK3). The samples blotted with actin antibody served as controls. B, HeLa cells were transfected as in A utilizing myc-tagged β-catenin and GST-tagged MLK3-WT or K/A followed by Western blot analysis with antibodies against myc (for ectopic β-catenin), β-catenin (ectopic and endogenous β-catenin), GST (for ectopic MLK3), and GAPDH (as control). C, HeLa cells were transfected as in B followed by Western blot analyses with the samples utilizing the antibodies indicated. The cells in lane 3 were harvested after a pretreatment with 500 nm pan-MLK inhibitor CEP11004 for 20 h. D, HeLa cells were transiently transfected with either control-siRNA or MLK3-siRNA for the indicated periods of time followed by Western blot analysis. E, total cell extracts from control MCF-7 and ZR75–1 breast cancer cells (first and third lanes, respectively) or their stable transfectants expressing MLK3shRNA (second and fourth lanes, respectively) were analyzed by Western blots with the antibodies indicated.