Fig. 4.

ATP9A co-localises with MON2 and DOPEY2 on SNX3-retromer endosomes. a ATP9A-HA is found on early endosomes and at the TGN. HeLa cells were transiently transfected with ATP9A-HA and fixed and stained with an anti-HA (green) antibody and antibodies detecting either endogenous TGN46 or EEA1 (red). The blue staining indicates DAPI. Scale bar represents 10 μm. b, c ATP9A-HA colocalises on endosomes labelled for the SNX3-retromer components SNX3 and VPS26. HeLa cells were transiently transfected with ATP9A and subsequently fixed and stained for VPS26 and SNX3. Arrows indicate endosomes co-labelled. The blue staining indicates DAPI. Scale bar represents 10 μm. d Quantification of the co-localisation between ATP9A and SNX3 (Pearsons 0.37 ± 0.08, Overlap Coefficient 0.58 ± 0.11, n = 15 technical replicates). Error bars indicate s.e.m. e HeLa cells were transiently transfected with ATP9A-HA, DOPEY2-GFP and MON2-FLAG and subsequently fixed and stained with anti-HA and anti-FLAG antibodies. Arrows indicate endosomes labelled for all three components. Scale bar represents 10 μm. f Quantification of the co-localisation between MON2 and DOPEY2 (Pearsons 0.57 ± 0.12, Overlap Coefficient 0.92 ± 0.08, n = 18 technical replicates), MON2 and ATP9A (Pearsons 0.49 ± 0.15, Overlap Coefficient 0.81 ± 0.17, n = 17 technical replicates) and DOPEY2 and ATP9A (Pearsons 0.45 ± 0.12, Overlap Coefficient 0.81 ± 0.11, n = 18). Error bars indicate s.e.m. g HeLa cells were transiently transfected with ATP9A and WLS-mCherry and subsequently fixed and stained for endogenous SNX3 and HA. Arrows indicate endosomes labelled for all three components. Scale bar represents 10 μm. h Representative blot showing DOPEY2-GFP binds both MON2 and ATP9A-HA. Cell extracts derived from HEK293 cells transiently transfected with GFP and ATP9A-HA or DOPEY2-GFP and ATP9A-HA were subjected to a GFP nanotrap and subsequently blotted with antibodies raised against HA and MON2