polyclonal anti glua1 antibody  (Alomone Labs)

Journal: Molecular Brain

Article Title: Postsynaptic density protein 95 (PSD-95) is transported by KIF5 to dendritic regions

doi: 10.1186/s13041-019-0520-x

Figure Lengend Snippet: ADPDZ3 expression reduces surface glutamate receptor 1 (GluA1) in dendrites. Cultured rat hippocampal neurons were infected with Sindbis viruses encoding GFP or GFP-ADPDZ3 and incubated for 9 h to allow expression. The cultures were then subjected to immunostaining using polyclonal GluA1 antibody and Cy3-conjugated anti-mouse IgG antibody. They were visualized using confocal microscopy. a , b Representative images of expressed neurons and selected dendrites in the analyses. Dotted boxes indicate analyzed dendrites. Scale bar: 20 μm. c , d ADPDZ3 expression reduced the number of surface GluA1 particles (GFP: 100.00% ± 7.83%, n = 18, 1486 μm; ADPDZ3: 66.50% ± 6.21%, n = 14, 1254 μm; ** P

Article Snippet: The cultures were pretreated using the preblock solution (2% BSA, 0.08 TritonX-100 in PBS) for 1 h and each antibody was directly added to the preblock solution for 2 h. The following antibodies were used for staining, each at a dilution of 1:50; monoclonal anti-PSD-95 antibody (clone 6G6-1C9; Affinity Bioreagents, Golden, CO, USA), polyclonal anti-PSD-95 antibody (Cell Signaling, Danvers, MA, USA), monoclonal anti-kinesin antibody (Clone: H2; Millipore, Temecula, CA, USA), polyclonal anti-synapsin I antibody (Millipore), polyclonal anti-GluA1 antibody (Upstate, Lake Placid, NY), polyclonal anti-GluA1 antibody (Alomone Labs, Jerusalem, Israel) for surface GluA1.The following antibodies were used for secondary staining, each at a dilution of 1:200: Alexa Fluor® 488 anti-rabbit IgG antibody (Molecular Probes, Eugene, OR, USA), Cy3-conjugated anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), Cy3-conjugated anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories), and Alexa Fluor® 647 anti-rabbit IgG antibody (Molecular Probes).

Techniques: Expressing, Cell Culture, Infection, Incubation, Immunostaining, Confocal Microscopy

Journal: Molecular Brain

Article Title: Postsynaptic density protein 95 (PSD-95) is transported by KIF5 to dendritic regions

doi: 10.1186/s13041-019-0520-x

Figure Lengend Snippet: Complexes of PSD-95-KIF5 colocalized with GluA1 particles in dendrites. Cultured hippocampal neurons were transfected with PSD-95-GFP constructs and incubated for days. The cultures were immunostained with monoclonal anti-PSD-95 antibody and polyclonal GluA1 antibody; they were subsequently stained with C3-conjugated anti-mouse IgG and Alexa−Fluor® 647 anti-rabbit IgG antibody. a Representative images of immunostaining in the first row. Each image was merged to show colocalization in the second row. A colocalized image of PSD-95 and KIF5 was collated with the image of GluA1. The colocalized points appeared white. Scale bar: 20 μm. b Boxed dendrites were enlarged to see the colocalization of GluA1 with the complex of PSD-95-KIF5A. c Staufen expression increased the association of PSD-95 and KIF5A. HA-PSD-95 and FLAG- KIF5A were cotransfected with 1, 2, or 3 μg of Myc-Staufen, or without Myc-Staufen as a control. After immunoprecipitation using anti-FLAG antibody or mouse IgG, immunoprecipitates were analyzed by Western blotting using anti-HA antibody. The lower panel shows expression of each group. d Quantified data of Western blot analyses (0 μg of Staufen: 100.0% ± 0.00%, n = 3; 1 μg of Staufen: 141.6% ± 16.96%, n = 3; 2 μg of Staufen: 192.3% ± 6.59%; 3 μg of Staufen; 274.4% ± 42.30%, n = 3). N values indicate the number of independent experiments. e Schematic diagram showing GluA1-containing vesicle transport mediated by PSD-95-KIF5A complex in dendrites. TARP: transmembrane AMPA receptor regulatory protein

Article Snippet: The cultures were pretreated using the preblock solution (2% BSA, 0.08 TritonX-100 in PBS) for 1 h and each antibody was directly added to the preblock solution for 2 h. The following antibodies were used for staining, each at a dilution of 1:50; monoclonal anti-PSD-95 antibody (clone 6G6-1C9; Affinity Bioreagents, Golden, CO, USA), polyclonal anti-PSD-95 antibody (Cell Signaling, Danvers, MA, USA), monoclonal anti-kinesin antibody (Clone: H2; Millipore, Temecula, CA, USA), polyclonal anti-synapsin I antibody (Millipore), polyclonal anti-GluA1 antibody (Upstate, Lake Placid, NY), polyclonal anti-GluA1 antibody (Alomone Labs, Jerusalem, Israel) for surface GluA1.The following antibodies were used for secondary staining, each at a dilution of 1:200: Alexa Fluor® 488 anti-rabbit IgG antibody (Molecular Probes, Eugene, OR, USA), Cy3-conjugated anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), Cy3-conjugated anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories), and Alexa Fluor® 647 anti-rabbit IgG antibody (Molecular Probes).

Techniques: Cell Culture, Transfection, Construct, Incubation, Staining, Immunostaining, Expressing, Immunoprecipitation, Western Blot