Quantification of fluorescence images was performed using Scion Image and ImageJ software. For the endocytosis
assay, internalization of GABAAR was monitored as described elsewhere by Goodkin et al. (2005) and Heisler et al. (2011). Cultures of hippocampal neurons (14–18 days in vitro) were incubated at selleck chemicals 4°C for 1 hr in the presence of an anti-GABAARβ2/3 antibody (clone 62-3G1). After incubation, the neurons were washed with PBS and then incubated in antibody-free medium to allow antibody-bound receptors to undergo internalization at 37°C for 90 min (GABAAR internalization was maximal at this time point), followed by fixation for 15 min with 4% paraformaldehyde. After fixation, neurons were blocked with 5% BSA for 30 min, CP-673451 ic50 exposed to the first of two secondary antibodies (20 μg/ml Alexa Fluor 488-conjugated goat anti-mouse) for 2 hr under
a nonpermeabilized condition, and then permeabilized by treatment with 0.25% Triton X-100 for 10 min, followed by incubation with the other secondary antibody (10 μg/ml Alexa Fluor 568-conjugated goat anti-mouse) for 1 hr. Observation was carried out under a Zeiss LSM510 confocal laser-scanning microscope. Red signals represented internalized surface receptor, and green signals represented receptors that remained on the cell surface. Full-length cDNA clones of KIF5s had been previously obtained by Kanai et al. (2000). Yeast two-hybrid assays were performed as described elsewhere by Setou et al. (2000). The detailed procedure is provided in the Supplemental Experimental Procedures. To perform live imaging of GFP-tagged GABAAR transport, after 7 days of culture, hippocampal neurons were transfected with α1, β3, and GFP-γ2 constructs (Twelvetrees et al., 2010). At 36–48 hr posttransfection, live neurons were observed under a Zeiss LSM710 Duo confocal laser-scanning microscope. Movement of GABAAR vesicles along dendrites was monitored over time, and images were acquired every second. Determination of the velocities was performed for 30 s. The path of individual below vesicles
was traced, and distances were evaluated using LSM710 software. Analysis and graphical representation were performed using ImageJ software and GraphPad Prism (GraphPad Software, San Diego, CA, USA). To monitor post-Golgi release of GABAAR vesicles, GABAAR α1, β3, and GFP-γ2 constructs were initially expressed in hippocampal cells in the presence of 10 μg/ml BFA (Wako Pure Chemical Industries, Osaka, Japan). After extensive BFA washout, cells were observed under an LSM710 confocal laser-scanning microscope. Quantification was performed as described elsewhere (Yin et al., 2012). At 1 hr after BFA washout, three regions of interest were drawn at peripheral locations, and the corresponding mean fluorescence represented an estimation of GFP-γ2 at non-Golgi locations. Golgi-associated fluorescence was determined from perinuclear regions.