Fig 2: GST-ATP1a1-M4M5 interacts directly with Rab27a.GST or GST-ATP1a1-M4M5 was incubated with A) melan-INK4a cell lysates, B) his6-Rab27aWT or his6-Rab5aWT. GST was immobilised on glutathione Sepharose. Co-precipitation was assessed by immunoblotting for Rab27a or his6 and GST. Blots are representative of 3 independent experiments.

Fig 3: Rab38 localisation to melanosomes is unaffected following ATP1a1 depletion.Melan-INK4a cells were treated with either NT or ATP1a1 siRNA and cultured for 72 h. Cells were fixed with PFA, permeabilised and immunolabelled with antibodies to Rab38 or ATP1a1. Phase contrast panels show melanosome distribution (A, D, G, J), immunostaining is shown for Rab38 (B and E) and ATP1a1 (H and K). In the merge panels (C, F, I, L) the pigment is inverted and pseudo-coloured red to aid co-localisation with the green immunoflrescence signals. Insets are a higher magnification of the boxed area. Arrows indicate co-localisation between pigment and Rab38 (C and F). Scale bar represents 10 µm.

Fig 4: Rab5a-positive vesicle distribution is unaffected following ATP1a1 depletion.Melan-INK4a cells were treated with either NT or ATP1a1 siRNA and cultured for 72 h. Cells were fixed with PFA, permeabilised and immunolabelled with antibodies to Rab5a. Phase contrast panels show melanosome distribution (A and C), panels B and D show Rab5a staining. Scale bar represents 10 µm.

Fig 5: ATP1a1 depletion disrupts endogenous Rab27a targeting to melanosomes.A) Melan-INK4a cells were plated on coverslips and treated with NT (A–C), Mlph (D–F), ATP1a1 (G–I), R3G (J–L) or ATP1a1+R3G (M–O) siRNA for 72 h before repeating the treatment. After a total of 7 days, cells were fixed with PFA, permeabilised and immunolabelled with an antibody to Rab27a. Phase contrast panels show melanosome distribution (A, D, G, J, M); panels B, E, H, K, N show Rab27a localisation. In the merge panels (C, F, I, L, O) the pigment is inverted and pseudo-coloured red to aid co-localisation with the green Rab27a signal. Insets are a higher magnification of the boxed area. Arrows indicate co-localisation. Scale bar represents 10 µm. B) Melan-INK4a cells were treated with NT, Mlph, R3G or ATP1a1 siRNA and cultured for 3 days. The PNS was then separated into the insoluble (I) and soluble (S) fraction by centrifugation at 100,000×g. Rab27a and Tubulin partitioning were assessed by immunoblotting with the antibodies indicated.