Computational protocol: A Role for Na+,K+-ATPase α1 in Regulating Rab27a Localisation on Melanosomes

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Protocol publication

[…] All tissue culture material was purchased from Invitrogen unless otherwise specified. Mouse WT melanocytes (melan-INK4a) and melan-ink4a-ashen (melan-ash2) were maintained in RPMI 1640 (+L-Glutamine) supplemented with 10% fetal calf serum, 100 U/mL penicillin, 100 U/mL streptomycin, 200 nM Phorbol-1,2-myristate-1,3-acetate (PMA; Calbiochem) and 200 pM Cholera Toxin (CT; Sigma) (Complete RPMI medium). All cells were cultured at 37°C under a humidified atmosphere containing 10% CO2. Cells and melanosome distribution was assessed using a light microscope. For immunofluorescence experiments, cells were grown on glass coverslips in 24-well plates. Transfection of plasmid DNA was performed using FuGENE6 (Roche Diagnostics) in a 1∶3 ratio of µg of DNA to µl of FuGENE6, according to the manufacturer's recommendations. For each well of a 24-well plate, 0.5 µg of DNA was mixed with 1.5 µl of FuGENE6 in 25 µl of serum-free medium OptiMEM. Following a 5 h incubation, the medium was replaced with RPMI (+L-Glutamine) supplemented with 10% (v/v) FBS. After overnight culture the medium was replaced with complete RPMI medium. For siRNA analyses, cells were transfected with siRNA to a final concentration of 100 nM using Oligofectamine (Invitrogen) according to the manufacturer's instructions. Following 5 h incubation the medium was removed and replaced with complete RPMI medium. For optimal siRNA depletion the siRNA treatment was sometimes repeated after 72 h. For membrane/cytosol fractionation experiments soluble (S100) and insoluble (P100) fractions were separated by centrifugation at 100'000×g of the PNS. The P100 was then resuspended in the same volume of loading buffer as the total volume of S100. Equal volumes of S100 and P100, relative to the protein concentration of the PNS for each siRNA treatment, were loaded on the gel. Quantification of ECL signals was carried out using ImageJ software. [...] Cells plated on coverslips were washed in PBS and fixed with 4% (w/v) paraformaldehyde (PFA) in PBS for 20 min. They were washed twice in PBS and once with 50 mM NH4Cl in PBS, and then permeabilised in PBS containing 2% FBS and 0.05% Saponin (PFS). Permeabilised cells were incubated with primary antibody diluted in PFS, washed three times in PFS and incubated with the appropriate fluorescently-conjugated secondary antibody (Molecular Probes) diluted 1∶400 in PFS. The coverslips were washed three times in PFS, twice in PBS and once in H2O, then mounted on microscope slides with either ImmunO-fluore mounting medium (MP Biomedicals) or Prolong Gold antifade reagent (Invitrogen). Microscopy was performed in the Facility for Imaging Light Microscopy (FILM) at Imperial College London. Immunofluorescence images were acquired at room temperature on a Zeiss AxioVert 200M laser scanning inverted confocal microscope ((LSM)-510) using Plan-Apochromat 63× 1.40 Oil DiC M27 objective, Zeiss AxioCam HRm camera and LSM 510 acquisition software. Images were processed with LSM Image Browser and Adobe Photoshop 8.0 software before compiling in Adobe Illustrator. […]

Pipeline specifications

Software tools ImageJ, Adobe Illustrator
Applications Miscellaneous, Laser scanning microscopy, Microscopic phenotype analysis
Chemicals Adenosine Triphosphate, Guanosine Diphosphate, Guanosine Triphosphate, Nucleotides, Potassium, Sodium