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. 2016 Jul 18;10:195–203. doi: 10.1016/j.ebiom.2016.07.020

Fig. 3.

Fig. 3

Nedd4L interacts with LPA1 on serine 319 residue. a. MLE12 cells were transfected with HA-Nedd4 or HA-Nedd4L plasmid for 48 h. Cell lysates were subjected to immunoprecipitation with a LPA1 antibody, followed by HA and LPA1 immunoblotting. Input lysates were analyzed by immunoblotting with HA and β-actin antibodies. b. MLE12 cells were transfected LPA1-V5 with or without HA-NEDD4L plasmid. Cell lysates were subjected to immunoprecipitation with a HA antibody, followed by V5 and HA immunoblotting. Input lysates were analyzed by V5, HA, and β-actin antibodies. c. MLE12 cells grown on glass bottom dishes were co-transfected with LPA1-V5 and HA-NEDD4L plasmid for 48 h, and then cells were fixed and immunostained with V5 and HA antibodies. LPA1-V5, green; HA-Nedd4L, red; nuclei, blue. d. MLE12 cells were treated with LPA (5 μM) for 30 min, and then cell lysates were subjected to immunoprecipitation with a LPA1 antibody, followed by Nedd4L and LPA1 immunoblotting. Input lysates were analyzed by Nedd4L immunoblotting. e. MLE12 cells were transfected with LPA1-V5 or LPA1S319A-V5 plasmid for 48 h. Cell lysates were subjected to immunoprecipitation with a V5 antibody, followed by Nedd4L immunoblotting. Input lysates were analyzed by immunoblotting with Nedd4L and β-actin antibodies. f. MLE12 cells were transfected with LPA1-V5, LPA1S319A-V5, or LPA1S331A-V5, with or without HA-NEDD4L plasmids for 48 h. Cell lysates were analyzed by V5, HA, and β-actin antibodies. Representative immunoblots were from at least three independent times.