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. 2017 Jun 13;8:15835. doi: 10.1038/ncomms15835

Figure 1. RAB6 associates with melanosomes.

Figure 1

(a,b) IFM on MNT-1 cells (a) and Normal Mouse Melanocytes (NMM) (b) stained for endogenous RAB6, TYRP1 and TGN46 (a) or Giantin (b), showing RAB6 distribution to the Golgi area (arrowheads) and in the vicinity of TYRP1- and melanin-positive melanosomes (white and red arrows respectively, 5X area). (c) Quantification of the number of endogenous RAB6 fluorescent structures associated with melanosomes in MNT-1, NMM and NHEM. (d) Live imaging frames of MNT-1 (n) cells (imaging plane Z-1) expressing GFP-RAB6 and mCh-VAMP7 (Pearson’s correlation coefficient; n=66, four independent experiments). (e) Live imaging frames of MNT-1 cells co-expressing GFP-TYRP1 and mCh-RAB6 with corresponding intensity profile of RAB6 across melanosomes measured along white line (right panel). (f) Representative electron micrograph of ultrathin cryosection of GFP-RAB6 expressing MNT-1 cells showed RAB6 association (anti-GFP, × 2 area) to Golgi Apparatus (GA, white arrows), vesicles (arrowheads) and limiting membranes of melanosomes (black arrows) positive for TYRP1. (g) Quantification of immunogold labelling for GFP-RAB6 relative to indicated cell compartments (230 gold particles; n=12 cells, 2 independent experiments). Data are normalized to the total number of gold particles and presented as means±s.e.m. (h) Immunogold labelling of RAB6 (PAG-10 nm) or GFP (arrows) of melanosome-enriched fractions from NHEM (left) or MNT-1 expressing GFP or GFP-RAB6 (PAG-15 nm, right). (i) Distribution of anti-GFP gold particles over stage-III/-IV purified melanosomes from MNT-1 expressing GFP-RAB6 presented as mean±s.e.m. (n=209 melanosomes, with at least 1 gold particle, quantified from 2 independent experiments). (j) Western Blot (WB) of PNS (1%, Post-nuclear supernatant) and melanosomes-enriched fraction (25%, Mel) isolated from NHEM or MNT-1 cells expressing or not GFP-RAB6 and probed with RAB6 or GFP (*), TYRP1, TYRP2, MART-1, GM130 or GAPDH antibodies. Blots are representative of 3 independent experiments. Scale bars: 10 μm (a,b and d,e), 500 nm (f–h), M.W.in kDa, two-tailed, Unpaired t test, ****P<0.0001.