Figure 7

Immunolocalization of Gau protein. Immunolocalization of Gau protein (green) in HUVEC cells (A, B). In (C, D), localization of MitoTracker Red CMXRos, a mitochondrial marker (red). In (E, F), colocalization of Gau protein (green) and mitochondria (red). After reaching confluence, human umbilical vein endothelial cells (HUVEC) were incubated with MitoTracker Red CMXRos (Invitrogen, France). Mitotracker Red CMXRos (200 nM) was added to the cells for 10 min. After this incubation, the cells were washed for 10 min with HBSS and then fixed directly with 4% PFA for 3 min at 4°C. The cells were covered with Vectashield mounting medium to avoid bleaching the fluorescence. All steps were performed in the dark. For immunodetection of Gau proteins, the mouse monoclonal antibody developed by ProteoGenix (Oberhausbergen, France) was used. To minimize any non-specific antibody binding, the cells were first incubated in 10% normal goat serum in PBS (0.1 M) containing 0.1% Triton X-100 and 2% bovine serum albumin (PBS buffer) for 1 h at room temperature. Then, they were incubated overnight at 4°C in the primary monoclonal antibody against Gau, which was diluted at 1/100 in a PBS (0.1 M) solution containing 10% normal goat serum and 2% bovine serum albumin. After being rinsed three times, the cells were incubated for 1 h with the secondary antibody, Alexa 488-coupled anti-mouse IgG (Invitrogen) diluted at 1:400. Finally, the cells were rinsed in PBS and mounted in a medium containing an antifading agent (Gel/MountR, Bibmeda, USA). The primary antibody was omitted for the controls. Images were acquired using the Zeiss LSM 710 NLO confocal microscope (63x objective, numerical aperture 1.35). All parameters (laser percentage and voltage, light, gain, exposure, offset values) were adjusted to achieve the best results.