Journal: The Journal of Neuroscience

Article Title: Subunit-Dependent Surface Mobility and Localization of NMDA Receptors in Hippocampal Neurons Measured Using Nanobody Probes

doi: 10.1523/JNEUROSCI.2014-22.2023

Figure Lengend Snippet: Surface localization of GFP-GluN-containing NMDARs in hippocampal neurons measured by dSTORM using the nanoGFP-AF647 probe. A , Representative images of rat hippocampal neurons (DIV14) infected with GFP-GluN3A subunit; total (labeled using primary anti-GFP antibody and secondary anti-antibody conjugated with AF488) and surface (obtained using 1000-fold diluted nanoGFP-AF647 probe) signals are shown; negative control was labeled without addition of nanoGFP-AF647 probe. B , Summary of relative surface expression signals obtained by labeling with differently diluted nanoGFP-AF647 probes (1,000×; 3,000×; 10,000×) as described in A ; measured in 10 μm segments of secondary or tertiary dendrites of rat hippocampal neurons infected with the GFP-GluN3A subunit ( n ≥ 24 segments in ≥6 different cells/group). C , Summary of relative surface expression of indicated GFP-GluN subunits measured in 10 μm segments of secondary or tertiary dendrites of infected rat hippocampal neurons, labeled as described in A ( n ≥ 24 segments in ≥6 different cells/group); one-way ANOVA ( F (3,287) = 53.0405, p < 0.001 followed by Bonferroni's multiple-comparisons test with p -values denoted in the figure. D , Selected dSTORM images of rat hippocampal neurons infected with GFP-GluN2A, GFP-GluN2B, or GFP-GluN3A subunits that were labeled with nanoGFP-AF647 probe. E , Histogram of the localization density of NMDARs containing the GFP-GluN2A, GFP-GluN2B, and GFP-GluN3A subunits labeled with the nanoGFP-AF647 probe. The AF647 localizations are plotted against distance to the edge of the synaptic region, measured in rat hippocampal neurons coinfected with tdTomato-Homer1c ( n > 161,793 nanoGFP-AF647 localizations/group). F , Histogram of the localization density of AF647 localizations plotted against distance to the edge of the synaptic region, measured in hippocampal neurons from cKO-GluN2A/GluN2B mice coinfected with GFP-GluN3A subunit and tdTomato-Homer1c (–Cre/GFP-GluN3A) or Cre-tdTomato-Homer1c (+Cre/GFP-GluN3A; n > 12,998 nanoGFP-AF647 localizations/group).

Article Snippet: Samples were then fixed with 4% PFA containing 4% sucrose in PBS for 7 min, permeabilized with 0.25% Triton X-100 in PBS for 5 min, and labeled with primary rabbit anti-GFP antibody (1:1000; Merck) followed by goat anti-rabbit antibody conjugated with AF488 (1:1000; Thermo Fisher Scientific).

Techniques: Infection, Labeling, Negative Control, Expressing

Journal: BioMed Research International

Article Title: Identification of the Functional Domain of HPIV3 Matrix Protein Interacting with Nucleocapsid Protein

doi: 10.1155/2020/2616172

Figure Lengend Snippet: Interaction of N and M. (a, b) After 293T cells were transfected with the corresponding plasmids, the protein was immunoprecipitated with anti-HA (a) or anti-Myc (b). The protein level was detected by WB. (c) N-Myc and HA-M plasmids were used to transfect 293T cells alone or together, and the protein expression in cell lysate and VLP was detected by WB. (d) After cotransfecting HeLa cells with plasmids encoding N-Flag and HA-M, N was stained with anti-Flag and AF488-conjugated fluorescent secondary antibody, and M was stained with anti-HA and AF568-conjugated fluorescent secondary antibody. Immunofluorescence was observed under a microscope. Scale bar = 10 μ m.

Article Snippet: The N was stained with rabbit anti-Flag primary antibody (Proteintech) and AF488-conjugated goat anti-rabbit fluorescent secondary antibody (Thermo Fisher).

Techniques: Transfection, Immunoprecipitation, Expressing, Staining, Immunofluorescence, Microscopy