Thus, it will be of interest to assess whether inhibition of Yif1B could influence 5-HT1A receptor desensitization that follows administration of antidepressants as mentioned before

Thus, it will be of interest to assess whether inhibition of Yif1B could influence 5-HT1A receptor desensitization that follows administration of antidepressants as mentioned before. Yif1B is implicated in 5-HT1A trafficking in dendrites Our results show that Yif1B plays a major role in the targeting of 5-HT1A receptors to the neuronal dendrites. (2 l of a 20 mm answer) were thoroughly mixed before the addition of Lipofectamine 2000 (1.25 l/coverslip). All cells were used 48 h after transfection. GST pull-down assay. GST-tagged fusion proteins were produced in the BL21DE3 strain of (Stratagene). After induction at 28C with 0.5 mm isopropyl–d-thiogalactopyranoside, the bacterial pellet was sonicated in PBS, 0.1% Triton X-100, Cy3 NHS ester and protease inhibitors (Sigma) and centrifuged at Rabbit polyclonal to KATNB1 14,000 for 15 min. Supernatants were incubated overnight with glutathione-Sepharose beads (GE Healthcare) at 4C, then washed in PBS. Extracts from LLC-PK1 cells expressing Yif1B or from hippocampus and cerebellum dissected from Sprague Dawley rats were homogenized by sonication in buffer A (20 mm HEPES, pH 7.4, 150 mm NaCl, 2 mm EDTA, 0.5% NP-40, protease inhibitors, 100 mg of tissue/ml of buffer). After centrifugation at 15,000 for 10 min, 100C200 g of proteins from cell lysates or 1 mg of proteins from tissue homogenates were incubated with glutathione-Sepharose beads coupled to the different GST fusion proteins in buffer A made up of 2.5 mg/ml bovine serum albumin, overnight at 4C. After washing three Cy3 NHS ester times, the proteins retained around the beads were eluted in sample buffer (Laemmli, 1970) and loaded on SDS-PAGE gel, followed by blotting to PVDF membrane and incubation with Yif1B affinity-purified antiserum (1:500). Immunofluorescence. For brain section immunofluorescence, rats were deeply anesthetized with pentobarbital (60 mg/kg, i.p.) and perfused with 4% paraformaldehyde (PFA) in PBS. Brains were postfixed in the same fixative for 24 h, and sections (30 m) were obtained using a vibratome. For immunofluorescence on transfected cells or neurons, coverslips with Cy3 NHS ester attached cells were washed three times with PBS+ (PBS made up of 0.1 mm CaCl2 and 0.1 mm MgCl2) at 37C, and fixed for 15 min at 37C with PBS+ containing 4% paraformaldehyde and 4% sucrose. For brain slices and coverslips, immunofluorescence experiments were performed as explained previously (Bou-Grabot et al., 2004a,b). Immunofluorescence images were generated using a Leica TCS-400 laser scanning confocal microscope (100 oil-immersion lens) or a TCS SP2 AOBS laser scanning confocal microscope (63 oil-immersion lens). The percentage of colocalization was calculated on confocal images acquired with a pixel size of 100 nm (objective 63, zoom 2.5), without any saturation of the two labels, and was calculated with the colocalization threshold program of ImageJ (Costes et al., 2004). The total dendritic tree length was measured Cy3 NHS ester with the length measurement of the Lucia 4.7 software (Nikon). Quantification of dendrite fluorescence. Contrast and brightness of confocal images were chosen to ensure that all relevant pixels were within linear range and were maintained identical for all those measurements. For double-labeling experiments, pictures were generated using Adobe Photoshop 7.0. Fluorescence profiles along dendrites were generated using the Lucia 4.71 software (Nikon). For the comparison of 5-HT1A-eGFPR distribution, all neurons showing intact morphology along their longest dendrite, with unambiguous visual identification of the axon, were analyzed (one dendrite per neuron). The variability of distribution in individual neurons was eliminated by using the cumulated fluorescence profiles obtained for 20 neurons in each group. Results Identification of Yif1B as a 5-HT1AR C terminus-interacting protein using the yeast two-hybrid assay To identify proteins interacting specifically with 5-HT1AR, we performed a yeast two-hybrid screening of a rat hippocampus cDNA library using the 5-HT1AR cytoplasmic C terminus (residues 406C422) as bait. Five clones corresponded to amino acids 22C311 of a protein recently named Yif1B (accession number: “type”:”entrez-protein”,”attrs”:”text”:”NP_942029″,”term_id”:”62388883″,”term_text”:”NP_942029″NP_942029), and were scored B as a Predicted Biological Score (Wojcik et al., 2002). This protein, not yet characterized in mammals, appeared as the ortholog of the protein Yif1p, a 35.5 kDa transmembrane protein that was first described as a Yip1p-interacting factor (Matern et al., 2000). Yip1p is usually localized in the Golgi membrane and ER-derived anterograde transport COPII vesicles (Otte et al., 2001). Yif1p was shown to interact with Yip1p and with transport Rab GTPases, and to play a critical role in.