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, the Green Fluorescent Protein (GFP) in pGFP-NT-TOPO-TA vector
, Human fibroblasts were seeded in 35 mm diameter plates and transfected at 50% confluency using Lipofectamine reagent following manufacturer's instructions
, Briefly, 1 µg plasmid and 10 µl Lipofectamine were mixed for 30 min in OptiMEM-1 culture medium (Invitrogen) and then added to cells for 4 hours at 37°C. This transfection medium was then replaced by DMEM-10% FCS for 2 days before ?-tubulin immunolabelling
, Non specific sites were blocked in PBS with 5% FCS and 0.01% Triton X-100 for 90 min and incubated overnight with the mouse monoclonal antibody C11 directed against human ?-tubulin (Santa Cruz Biotechnology) or mouse monoclonal antibody against human ?-tubulin (Sigma Aldrich) diluted at 1:200 in blocking buffer. After washing, immunostaining was revealed by incubation for 2 hours with an anti-mouse antibody-Cy3 (Jackson ImmunoResearch) diluted at 1:200 in blocking buffer. For double immunolabelling, primary antibodies against human dynamin 2 (C18-goat antibody, Immunocytochemistry. Fibroblasts were washed in PBS and then fixed 20 min at -20°C in acetone before immunolabelling
, Immunostaining was revealed using a mixture of rabbit anti-goat-Cy3 and sheep anti-mouse
, Chemicon International) at 1:400 in blocking buffer. Labelled cells were visualized by fluorescence microscopy (Axiophot system Zeiss, Germany) coupled with a CCD camera and images were acquired using the Metaview software, FITC
, Non specific sites were blocked for 2 hours at room temperature in PBS with 5% nonfat dry milk and 0.1% Triton X-100. Incubation with primary antibody was performed overnight at 4°C for antibody against DNM2 (C18-goat polyclonal antibody raised against a peptide mapping at the carboxy terminus of DNM2, Western-blotting. Twenty µg of protein from control and CNM-fibroblasts in loading buffer (50 mM Tris-HCl, 2% SDS, 10% glycerol, vol.1
, Jackson ImmunoResearch and anti-mouse-HRP from Dako) diluted at 1:2000 in blocking buffer. Detection was performed using the Supersignal West Pico Chemiluminescent kit (Pierce)
, Human DNM2 mRNA GenBank NM_001005360. Human DNM2 genomic sequence GenBank NT_011295. Human DNM2 protein Genbank P50570. Mouse DNM2 protein Genbank P39054. Human DNM1 protein Genbank Q05193. Human DNM3 protein Genbank Q9UQ16. Zebrafish DNM protein Genbank AAH65325. Drosophila DNM protein Genbank P27619. Caenorhabditis elegans DNM protein Genbank P39055, Yeast DNM protein Genbank, p.54861