Activation of PAR1 initiates assembly of an ubiquitin-dependent TAB2–TAB1–p38 signaling complex on endosomes. (A and B) PAR1 WT or 0K mutant coexpressed with TAB2 WT tdTomato in HEK293 cells were stimulated with 100 µM SFLLRN. Images of live cells at 81 s are shown. Arrowheads show PAR1 WT and TAB2 WT containing punctae. PAR1 WT and TAB2 WT colocalization is indicated by black punctae in the merged image. Insets are magnifications of the boxed areas. Bars: (main panels) 10 µm; (insets) 2.5 µm. See Fig. S4 (A–D). (C) Quantification of TAB2 WT tdTomato colocalization with PAR1 WT (black lines) or 0K mutant (red lines) on endosomes induced by 100 µM SFLLRN. Solid lines represent the mean and the dashed lines represent the standard deviation. n > 5 cells. (D and E) PAR1 WT coexpressed with either TAB2 WT or CA mutant tdTomato in HEK293 cells were stimulated with 100 µM SFLLRN. Arrowheads show PAR1 WT and TAB2 WT positive punctae at 9 min. PAR1 WT and TAB WT containing punctae are indicated by black punctae in the merged image. Insets are magnifications of boxed areas. Bars: (main panels) 10 µm; (insets) 2.5 µm. (F) Quantification of PAR1 WT colocalization with either TAB2 WT (black lines) or CA mutant (red lines) tdTomato on endosomes induced by 100 µM SFLLRN. Solid lines represent the mean and the broken lines represent the standard deviation. n > 5 cells. (G) Endogenous PAR1 was immunoprecipitated from endothelial cells stimulated with 10 nM α-Th, and coassociated p38, TAB1, and TAB2 were determined. (H) Phosphorylated p38 was immunoprecipitated from PAR1 HeLa cells stimulated with 10 nM α-Th, and coprecipitated p38-α, TAB1, and TAB2 was detected. (I) PAR1-expressing HeLa cells coexpressing TAB2 WT or CA mutant were stimulated with 10 nM α-Th, and coassociated p38, TAB1, and TAB2 were determined.