Figure 3.

The effect of GDI1/2 knockout on the localization of Rab proteins.A, immunoblots showing the establishment of U2OS clonal cells harboring doxycycline (Dox)-inducible expression of V5-GDI1 in the absence of endogenous GDIs. In the top panel, the upper and lower bands represent GDI1 and GDI2, respectively. The specificity of the anti-GDI antibody used in the top panel was examined using parental U2OS cells (P) and U2OS GDI1 KO (1) or GDI2 KO (2) polyclonal cells. Four monoclonal GDI1/2 DKO cells (#2, #4, #5, and #28) were established. GAPDH was used as a loading control (bottom panel). B, post-nuclear supernatants of parental U2OS cells as well as U2OS GDI1/2 DKO cells treated without (−) or with (+) dox to induce the expression of V5-GDI1 were fractionated into the cytosol (C) and membrane (M) fractions. Representative immunoblots of three independent experiments with the indicated antibodies are shown. DJ-1 was used as a representative cytosolic protein, whereas Na+/K+ ATPase α-1 subunit was used as a membrane protein. C, the comparison of the ratio of cytosolic to total Rab proteins among Rab proteins in U2OS cells. D, the comparison of the ratio of cytosolic to total Rab proteins between U2OS cells, U2OS GDI1/2 DKO cells without dox (DKO), and U2OS GDI1/2 DKO cells with dox to induce the expression of V5-GDI1 (DKO+GDI1). The band intensity of the Rab proteins was quantified, and the ratio of cytosolic Rab to total Rab (cytosol + membrane) was calculated. The circles, the bars, and the error bars in the graphs represent individual values, the mean values, and their standard errors, respectively. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. 'n.s.' means 'not significant'. The p-values and statistical tests used are summarized in Table S1. DKO, double knockout; GDI, GDP-dissociation inhibitor.