Figure 3. The D620N mutation leads to reduced endosomal association of FKBP15 and the WASH complex.

(a,b) Cells expressing either WT or D620N GFP-VPS35 were treated with siRNA to silence expression of endogenous VPS35, fixed and labelled for GFP and FKBP15 (a) or FAM21 (b). Cells were imaged by fluorescence microscopy. Scale bar, 20 μm. (c) Cells stably expressing WT or D620N GFP-tagged VPS35 were treated with siRNA to abolish expression of endogenous VPS35 and subsequently fixed and labelled for GFP and either VPS26, FKBP15 or FAM21. The cells were imaged using Cellomics automated fluorescence microscopy. Approximately 250 cells per well in four wells were analysed for each cell line. The data for FKBP15 and FAM21 spot intensity were normalized to GFP-VPS35 and VPS26 signals. P<0.0002 for both FKBP15 and FAM21 spot intensity in D620N compared with WT. Error bars indicate s.d. (d) Cells stably expressing either WT GFP-VPS35 or the D620N mutant were treated with siRNA to silence the expression of endogenous VPS35. Cells were permeabilized by flash freezing followed by rapid thawing and centrifuged to separate supernatant (S) and membrane pellet (P) fractions. (e) The graph shows the percentage of membrane-associated FKBP15 and strumpellin and is the mean of three experiments. The error bars indicate s.e.m. A representative blot of three experiments is shown, indicating the efficacy of the knockdown of endogenous VPS35 and also further showing that membrane proteins such as the CIMPR are detected only in the pellet fraction.