Figure 4.

Rab8 GEF Rabin8 also interacts with and activates Rab8 in LPS stimulated Macrophages. (a) Immunoprecipitation of Rabin8 using anti-myc antibodies from cell lysates of ± LPS (30 min) RAW 264.7 Rab8a KO cells reconstituted with myc-Rab8a. Fluorescence microscopy images of fixed (b) ± LPS (30 min) RAW 264.7 cells transiently overexpressing GFP-Rabin8 or (c) LPS-treated cells co-expressing td-Tomato-Rab8a and GFP-Rabin8 and stained with Alexa350-phalloidin. Scale bars, 10 µm. (d) Immunoblotting for levels of GTP-Rab8 pulled-down from the lysate of control and Rabin8 KO cells ± LPS (100 ng/ml, 15 min) using GSH-Sepharoase beads with GST-OCRL-RBD. Levels of active Rab8 was quantified by using the densitometric ratio between the band intensities of captured Rab8 and GAPDH. Significance was measured via two-way analysis of variance (ANOVA) (*P < 0.05, n = 3)