Figure 6.

Fractionation of Rab proteins in the cytosol and membrane fractions using Triton X-114. Post-nuclear supernatants of HEK293A (A and B), A549 (C and D), and HEK293A cells overexpressing HA-Rab10 or HA-Rab29 (E and F) were fractionated into the cytosol and membrane fractions by ultracentrifugation. Each fraction was further fractionated using Triton X-114 into aqueous and detergent phases and subjected to immunoblotting with the indicated antibodies. DJ-1 was used as a representative hydrophilic protein, whereas the transferrin receptor was used as a hydrophobic protein. Note that, for this experiment, equal amounts of protein, not volumes of the samples, were loaded for the cytosol and membrane fractions. The amount of Rab10 in the membrane fraction seems larger than that in the cytosol fraction, but this does not reflect the actual ratio of the amounts of Rab10 in the cytosol and membrane fractions as shown in Figure 1. Also, note that the transferrin receptor was observed both in the aqueous and detergent phases only when the cells were first mechanically homogenized for the fractionation by ultracentrifugation (A, C, and E). The band intensity of the Rab proteins was quantified, and the ratio of Rab in the detergent phase to total Rab (aqueous + detergent) 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 < 0.001. The p-values and statistical tests used are summarized in Table S1.