Fig 4.

Active Rac1 coexpression enhances Net1A stability. (A) MCF7 cells were transfected with HA-Net1A alone or with Myc-V¹²Rac1. Two days later the cells were incubated with the protein synthesis inhibitor cycloheximide (CHX) for different lengths of time. After lysis, the amount of HA-Net1A was determined by Western blotting. Shown are results of a representative experiment. (B) Quantification of Net1A stability. Shown are the averages of three independent experiments. Error bars are standard errors of the means. (C) Addition of the proteasome inhibitor MG132 is not sufficient to alter Net1A localization. MCF7 cells were transfected with expression plasmids for HA-Net1A, alone or with Myc-V¹²Rac1. Cells were incubated with MG132 overnight prior to fixation and stained for HA-Net1A (green) and DNA (blue). (D) Quantification of the effects of MG132 on Net1A localization. Shown are the averages of three independent experiments. Error bars are standard errors of the means. *, P < 0.05; ***, P < 0.001.