MAP2K5 inhibition attenuates the formation of OPTN and ANG protein aggregates in mammalian cells. NIH3T3 cells were transiently transfected with wild-type GFP-OPTN or GFP-OPTN mutants (E50K or E478G) (A) and wild-type GFP-ANG or GFP-ANG mutants (K17I, K40I, or P112L) (B). At 36 h after transfection, cells were incubated with vehicle or 10 µM BIX 02189 (MAP2K5 inhibitor) for 12 h. Cells were lysed in NP40 lysis buffer, and the lysates were separated into soluble and insoluble fractions: (Sup.) supernatant; (Ppt.) precipitate. OPTN or ANG proteins in the cellular fractions were detected with Western blot analysis using an antibody against GFP. DMSO (0.1% v/v) was used as a vehicle. The densitometry analysis was plotted as an intensity ratio of soluble GFP-OPTN/tubulin (Sup.), insoluble GFP-OPTN/actin (Ppt.), and total GFP-OPTN/tubulin (Total). The densitometry analysis for GFP-ANG was done in the same manner. The results of the densitometric analysis (bottom) are presented as the mean ± SD (n = 3); (*) P < 0.05 versus vehicle.
