Journal: Scientific Reports
Article Title: Hyaluronic acid based extracellular matrix regulates surface expression of GluN2B containing NMDA receptors
doi: 10.1038/s41598-017-07003-3
Figure Lengend Snippet: ECM removal leads to increased surface expression of GluN2B in a β1 - integrin dependent manner. ( A ) Dissociated hippocampal cultures were treated with Hya over night and stained against the total amount of GluN2B and the dendritic marker Map2 (scale bar: 10 μm. ( B ) Total GluN2B expression is not affected by ECM removal (Dendrites: Ctl 1 ± 0.10, n = 30; Hya 0.89 ± 0.03, n = 30, P = 0.31; Synapses: Ctl: 1 ± 0.03, n = 30; Hya: 1.05 ± 0.03, n = 30, P = 0.27; average ± SEM; unpaired t-test). ( C ) Quantitative WB of lysed cortical cultures (DIV21) pretreated with Hya over night show no significant change in GluN2B immunoreactivity. ( D ) Dissociated hippocampal cultures at DIV21-24 were treated with Hya over night and stained against surface GluN2B (green) and the synaptic marker PSD-95 (scale bar: 10 μm). ( E ) Synaptic GluN2B surface expression at various time points after Hya treatment (Ctl: 1 ± 0.04, n = 24; Hya 1,5 h: 1.08 ± 0.04, n = 22, P = 0.76; Hya 3 h: 1.40 ± 0.09, n = 30, P = 0.0001; Hya 6 h: 1.41 ± 0.13, n = 9, P = 0.002; Hya 12 h: 1.35 ± 0.08, n = 8, P = 0.01; Hya 48 h: 1.18 ± 0.05, n = 8, P = 0.04 average ± SEM; One way-ANOVA, Dunnett’s Multiple Comparison Test). ( F,G ) GluN2B surface expression at synapses and dendrites increases after ECM degradation and can be restored by simultaneous application of the β1-integrin function blocking antibody CD29. ( F ) Synapses: Ctl: 1.0 ± 0.05, n = 68; Hya: 1.3 ± 0.05, n = 70; Hya + CD29: 0.93 ± 0.03, n = 51. ( G ) Dendrites: Ctl 1.00 ± 0.04, n = 36; Hya 1.78 ± 0.11, n = 35; Hya + CD29 0.96 ± 0.03, n = 34; average ± SEM; One-way ANOVA, P
Article Snippet: Antibodies and drugs The following commercial antibodies were used for Immunocytochemistry (ICC) and Western blot (WB) in the concentrations indicated: rabbit (rb) antibodies against GluN2B (alomone labs; ICC live staining: 1:200, fixed staining.
Techniques: Expressing, Staining, Marker, CTL Assay, Western Blot, Blocking Assay