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. 2019 Oct 24;8:e50471. doi: 10.7554/eLife.50471

Figure 6. RhoGDI extracts RhoGTPases from membranes in vitro.

(A) Experimental setup of in vitro experiments: prenylated RhoGTPases were reconstituted on supported lipid bilayers (SLBs) in flow chambers and imaged by TIRF. Wash off experiments were designed to avoid RhoGTPase rebinding to membranes and performed controlling the flow rate via a syringe pump; (B) TIRF imaging allows for selective imaging of RhoGTPases at the membrane. Prenylated and unprenylated Cdc42 were imaged in the same conditions and TIRF signal at membranes was quantified (n = 4 for each condition); (C) Wash off experiments: prenylated Cdc42 reconstituted on SLBs were washed with imaging buffer only (control), in presence of 5 μM RhoGDI or RabGTTase Beta. Time lapse images at selected time points and quantification of the full experiments are shown. Decay curves were fitted with a monoexponential function; (D) Comparison of the Koff values obtained by fitting the decay curves (n = 3 for control and RabGTTase Beta, n = 2 for RhoGDI); (E) Schematic representation of the proposed mode of action of the two RhoGTPases solubilizers. RabGGTase Beta sequesters RhoGTPases in solution, whereas RhoGDI actively extracts RhoGTPases from the membranes. Scale bar 10 μm. Unpaired student’s t-test, 2-tailed distribution, equal variance statistical analysis. **p<0.01, ***p<0.001, ****p<0.0001.

Figure 6—source data 1. RhoGDI extracts RhoGTPases from membranesin vitro.

Figure 6.

Figure 6—figure supplement 1. In vitro data analysis.

Figure 6—figure supplement 1.

(A) Raw data. Wash off experiments were started after the GTPase signal at the membrane was stable. After starting the syringe pump, a signal overshoot occurred; (B) The overshoot signal was used as a reference point to align different experiments and cut off for further analysis; (C) Data were background corrected and normalized to display multiple curve on the same graph; (D) Data were fitted with a monoexponential decay curve to obtain dissociation constants (Koff) of the GTPases from the membrane.
Figure 6—figure supplement 2. Purified RhoGDI protein.

Figure 6—figure supplement 2.

Coomassie stain of 12% SDS-PAGE to assess purity of RhoGDI purified from bacteria; lane 1: start, lane 2: flow through, lane 3: TEV cleavage, lane 4: tag removal, lane 5: end product after gel filtration.