glur1 subunit  (Alomone Labs)

Journal: Nature Communications

Article Title: Aβ-dependent reduction of NCAM2-mediated synaptic adhesion contributes to synapse loss in Alzheimer's disease

doi: 10.1038/ncomms9836

Figure Lengend Snippet: Disruption of NCAM2 functions at the neuronal cell surface promotes glutamatergic synapse disassembly. ( a – e ) Cultured hippocampal neurons were either mock-treated or incubated with the recombinant soluble extracellular domains of NCAM2 (NCAM2-ED), antibodies against the extracellular domain of NCAM2 (NCAM2mAb), or Aβ 1-42 oligomers. In a , b , neurons were labelled with antibodies against the extracellular domain of GluR1 before permeabilization of membranes with detergent, and co-labelled with antibodies against synaptophysin after permeabilization of membranes with detergent. Representative images of dendrites are shown ( a ). Note co-localization of cell surface GluR1 accumulations with synaptophysin clusters in mock-treated neurons, and increased levels of non-synaptic cell surface GluR1 accumulations in neurons treated with NCAM2-ED, NCAM2mAb or Aβ 1-42 . Graphs ( b ) show the percentage of synaptic and non-synaptic GluR1 clusters relative to total number of GluR1 clusters along dendrites and numbers of synaptophysin accumulations per dendrite length (mean+s.e.m.). * P

Article Snippet: Goat antibodies against the extracellular domain of CHL1 (AF2147; R & D systems) were used for WB (1 μg ml−1 ); goat polyclonal and mouse monoclonal antibodies against synaptophysin (sc-7568, sc-17750; Santa Cruz Biotechnology) were used for WB (1 μg ml−1 ) and IC (4 μg ml−1 ); mouse monoclonal antibodies against actin (sc-8432; Santa Cruz Biotechnology; 1 μg ml−1 ), VGLUT1 (sc-377425; Santa Cruz Biotechnology; 1 μg ml−1 ) and VGAT (sc-393373; Santa Cruz Biotechnology; 1 μg ml−1 ) were used for WB; mouse monoclonal antibodies against PSD95 (clone K28/86, Millipore) were used for WB (0.1 μg ml−1 ) and IC (1 μg ml−1 ); mouse monoclonal antibodies against MAP2 (M4403; Sigma; 1:100) were used for IC; mouse monoclonal antibodies against Aβ (sc-28365; Santa Cruz Biotechnology) were used for WB (1 μg ml−1 ), IC (2 μg ml−1 ), PL (2 μg ml−1 ); rabbit polyclonal antibodies against Aβ (pre-diluted Aβ42 detection antibody; KHB3441 ELISA kit; Life Technologies) were used for ELISA; human-specific mouse monoclonal antibodies against Aβ (6E10) (Covance) were used for IH (1:100) and WB (1:1,000); rabbit monoclonal antibodies against Aβ1-42 (D3E10, Cell Signaling Technology; 1:200) were used for WB; rabbit polyclonal (A5060) and mouse monoclonal antibodies against actin (Sigma) were used for WB (1:1,000); rabbit polyclonal antibodies against the extracellular epitope of the GluR1 subunit of AMPA receptors (AGC-004; Alomone Labs, Jerusalem, Israel) were used for IC (1:100); and rabbit polyclonal antibodies against the extracellular epitope of the NR1 subunit of NMDA receptors (AGC-001; Alomone Labs) were used for IC (1:100).

Techniques: Cell Culture, Incubation, Recombinant

Journal: Nature Communications

Article Title: Aβ-dependent reduction of NCAM2-mediated synaptic adhesion contributes to synapse loss in Alzheimer's disease

doi: 10.1038/ncomms9836

Figure Lengend Snippet: Aβ 1-42 reduces the number of GluR1-containing synapses in the NCAM2-dependent manner. ( a ) Representative images of dendrites of cultured hippocampal neurons transfected either with control negative miRNA (negative miR) or NCAM2miR and either mock-treated or incubated with Aβ 1-42 . Transfected neurons were identified by fluorescence of GFP, which is co-expressed together with miRNA. Neurons were co-labelled with antibodies against cell surface GluR1 and synaptophysin. Note that the number of synaptic GluR1 clusters is reduced and the number of non-synaptic GluR1 clusters is increased in neurons transfected with NCAM2miR. Scale bar, 10 μm. ( b , c ) Graphs show mean+s.e.m. percentage of synaptic and non-synaptic GluR1 clusters relative to the total number of GluR1 clusters along dendrites ( b ) and numbers of synaptophysin accumulations per dendrite length normalized to the mean number in mock-treated neurons ( c ) for neurons described in ( a ). ( d – f ) Graphs show mean+s.e.m. percentage of synaptic and non-synaptic GluR1 clusters relative to the total number of GluR1 clusters along dendrites ( d ), number of synaptophysin accumulations per dendrite length normalized to the mean number in mock-treated neurons ( e ), and area/length ratio ( f ) in cultured hippocampal neurons transfected either with GFP alone or co-transfected with GFP and non-mutated NCAM2 (NCAM2WT) or NCAM2D693A mutant and either mock-treated or incubated with Aβ 1-42 . ( g , h ) Graphs show mean+s.e.m. percentage of non-synaptic GluR1 clusters relative to the total number of GluR1 clusters along dendrites ( g ) and area/length ratio ( h ) in cultured hippocampal neurons co-transfected with NCAM2 miR and either GFP, non-mutated NCAM2 (WT) or NCAM2D693A mutant (D693A) and either mock-treated or incubated with Aβ 1-42 . In b – h , * P

Article Snippet: Goat antibodies against the extracellular domain of CHL1 (AF2147; R & D systems) were used for WB (1 μg ml−1 ); goat polyclonal and mouse monoclonal antibodies against synaptophysin (sc-7568, sc-17750; Santa Cruz Biotechnology) were used for WB (1 μg ml−1 ) and IC (4 μg ml−1 ); mouse monoclonal antibodies against actin (sc-8432; Santa Cruz Biotechnology; 1 μg ml−1 ), VGLUT1 (sc-377425; Santa Cruz Biotechnology; 1 μg ml−1 ) and VGAT (sc-393373; Santa Cruz Biotechnology; 1 μg ml−1 ) were used for WB; mouse monoclonal antibodies against PSD95 (clone K28/86, Millipore) were used for WB (0.1 μg ml−1 ) and IC (1 μg ml−1 ); mouse monoclonal antibodies against MAP2 (M4403; Sigma; 1:100) were used for IC; mouse monoclonal antibodies against Aβ (sc-28365; Santa Cruz Biotechnology) were used for WB (1 μg ml−1 ), IC (2 μg ml−1 ), PL (2 μg ml−1 ); rabbit polyclonal antibodies against Aβ (pre-diluted Aβ42 detection antibody; KHB3441 ELISA kit; Life Technologies) were used for ELISA; human-specific mouse monoclonal antibodies against Aβ (6E10) (Covance) were used for IH (1:100) and WB (1:1,000); rabbit monoclonal antibodies against Aβ1-42 (D3E10, Cell Signaling Technology; 1:200) were used for WB; rabbit polyclonal (A5060) and mouse monoclonal antibodies against actin (Sigma) were used for WB (1:1,000); rabbit polyclonal antibodies against the extracellular epitope of the GluR1 subunit of AMPA receptors (AGC-004; Alomone Labs, Jerusalem, Israel) were used for IC (1:100); and rabbit polyclonal antibodies against the extracellular epitope of the NR1 subunit of NMDA receptors (AGC-001; Alomone Labs) were used for IC (1:100).

Techniques: Cell Culture, Transfection, Incubation, Fluorescence, Mutagenesis