Fig. 5.

USP11 deubiquitinates and stabilizes LPA1. a. MLE12 cells were co-transfected with LPA1-V5 and USP11-V5 or USP5-V5 plasmids for 48 h, and cells were treated with LPA (5 μM) for 0–60 min. Cell lysates were analyzed by immunoblotting with V5 and β-actin antibodies. b. MLE12 cells were transfected with USP11-V5 plasmid for 48 h, and then cells were treated with LPA (5 μM) for 30 min. Cell lysates were subjected to immunoprecipitation with an ubiquitin antibody, followed by LPA1 immunoblotting. Input lysates were analyzed by immunoblotting with LPA1, V5, and β-actin antibodies. c. MLE12 cells grown on glass bottom dishes were co-transfected with Usp11-V5 and Lpa1-myc plasmids for 48 h. Cells were fixed and immunostained with V5 and myc antibodies. USP11-V5, green; LPA1-myc, red; nuclei, blue. d. MLE12 cells were transfected with LPA1-V5 plasmid for 48 h, and then cells were treated with LPA (5 μM) for 30 min. Cell lysates were subjected to immunoprecipitation with a V5 antibody, followed by USP11 immunoblotting. Input lysates were analyzed by immunoblotting with USP11 and V5 antibodies. Representative immunoblots were from at least three independent times. e. Scheme shows that LPA treatment switches LPA1-USP11 complex to LPA1-Nedd4L complex.