Journal: Cell reports

Article Title: Btbd11 supports cell-type-specific synaptic function

doi: 10.1016/j.celrep.2023.112591

Figure Lengend Snippet:

Article Snippet: Mouse anti Psd95 (clone K28/43) , NeuroMab , Cat# K28/43, RRID:AB_2877189.

Techniques: Binding Assay, Control, Virus, Recombinant, Titration, Plasmid Preparation, Electron Microscopy, Mass Spectrometry, Mutagenesis, Sequencing, Cloning, Variant Assay, Software, Microscopy

Journal: Research Square

Article Title: Synaptogenesis by Cholinergic Stimulation of Astrocytes

doi: 10.21203/rs.3.rs-2566078/v1

Figure Lengend Snippet: Hippocampal neurons plated in glass coverslips were co-cultured for 24 hours with astrocytes plated in 24-well plates and pre-stimulated with increasing concentrations of carbachol (0, 0.01, 0.10, and 1 mM). At the end incubations, protein from neuronal cultures were extracted and Western blots were run for synaptophysin and PSD-95; synaptophysin and PSD-95 protein levels were normalized to total protein content determined after Coomassie blue stain of the membranes. (A) Representative immunoblots for synaptophysin (upper left) and PSD-95 (upper right); lower images: representative loading control membranes after Coomassie blue staining. (B)Densitometric quantification of synaptophysin and PSD-95 bands normalized to total protein content and expressed as fold over control. Control is represented as dotted line. Shown are means ± SEM. Statistical analysis was performed using one-way ANOVA followed by a Dunnett’s Multiple Comparison test (***: p < 0.001); n = 5 coverslips for 10 and 100μM carbachol; n = 14–15 coverslips for control and 1 mM carbachol.

Article Snippet: The mouse monoclonal antibodies PSD95 (Clone K28/43) for immunoblotting, and NR2B (Clone N59–36) were developed by and obtained from the UC Davis/NIH Neuromab facility.

Techniques: Cell Culture, Western Blot, Staining, Control, Comparison

Journal: Research Square

Article Title: Synaptogenesis by Cholinergic Stimulation of Astrocytes

doi: 10.21203/rs.3.rs-2566078/v1

Figure Lengend Snippet: (A-C): Primary hippocampal neurons were treated with TSP1 (5, 10 μg/mL) for 24 hours. Immunocytochemical labeling of the pre and postsynaptic proteins synaptophysin and PSD-95 was performed. Synaptophysin ( A ), PSD-95 ( B ) and overlapping pre and postsynaptic puncta (indicative of structural synapses) ( C ) were quantified and the distribution of the data is shown (the bold, dashed line represents the median; the upper and lower dotted lines represent the 75 th and 25 th percentile, respectively; n= 14–20 neurons from 5–6 coverslips). Statistical comparison of the means was performed using one-way ANOVA followed by Dunnett’s ad hoc test: *: p < 0.05 vs. control. ( D ): Astrocytes were treated for 24 hours with 1 mM carbachol or left untreated; after carbachol treatment washout astrocytes were co-cultured with hippocampal neurons for 24 hours in the presence or absence of gabapentin (15, 30 μM) to block TSP1 function in neurons. Presynaptic (synaptophysin) and postsynaptic (PSD95) puncta overlap was quantified as previously described. The distribution of the data is shown (the bold, dashed line represents the median; the upper and lower dotted lines represent the 75 th and 25 th percentile, respectively; n= 25–43 neurons from 4 independent experiments). Statistical comparison of the means was performed using one-way ANOVA followed by Bonferroni’s Multiple Comparison test. **: p < 0.01 vs. control; ##: p < 0.01, ###: p < 0.001 vs. carbachol. (A-D): The bold, dashed line represents the median; the upper and lower dotted lines represent the 75 th and 25 th percentile, respectively.

Article Snippet: The mouse monoclonal antibodies PSD95 (Clone K28/43) for immunoblotting, and NR2B (Clone N59–36) were developed by and obtained from the UC Davis/NIH Neuromab facility.

Techniques: Labeling, Comparison, Control, Cell Culture, Blocking Assay