A sex difference in the response of the rodent postsynaptic density to synGAP haploinsufficiency
- Caltech, United States
- University of Edinburgh, United Kingdom
- Centre for Brain Development and Repair, India
Figures
Figure 1
Generation of synGAP null rats.
(A) SynGAP targeting strategy introduces a frame shift mutation into exon 8. (B) Quantification of synGAP immunoblots (inset) of cortical homogenates was performed as described in Materials and metho…
Figure 2 with 1 supplement
Averaged ratios of synGAP and TARPs to PSD-95 in PSDs from WT and HET Rats and Mice.
PSDs were purified from the brains of individual animals as described under Materials and methods. The ratios of synGAP to PSD-95 (A) and TARPs to PSD-95 (B) were determined as described under …
Figure 2—figure supplement 1
Measurement of densities and calculation of ratios.
(A) Example of an immunoblot that was double-stained for synGAP and PSD-95 as described under Materials and methods. (B) Example of an immunoblot that was double-stained for TARPs and PSD-95 as …
Figure 3 with 3 supplements
Correlation of the ratios TARPs/PSD-95 and synGAP/PSD-95 among individual animals.
Each point represents mean ratios for a single animal. Corrected ratios and Spearman’s rank correlation coefficients were determined as described under Materials and methods. (A) All animals, …
Figure 3—figure supplement 1
Intensity of PSD-95 bands on immunoblots.
Blots were double-stained for PSD-95 and the target proteins as described under Materials and methods. SynGAP, GluN2B, NLG-1 and NLG-2 were fractionated on 8% gels. TARPS were fractionated on 12% …
Figure 3—figure supplement 2
Correlation analysis between synGAP/PSD-95 and TARP/PSD-95 for data from 7 and 12 week old female mice and rats.
Data was acquired as described under Materials and methods and in Figure 3. The small individual data sets indicated in the headings of each panel were normal in distribution. Therefore, Pearson’s …
Figure 3—figure supplement 3
Correlation analysis between synGAP/PSD-95 and TARP/PSD-95 for data from 7 and 12 week old male mice and rats.
Data was acquired and analyzed as described for Figure 3—figure supplement 2. None of the data sets containing only males showed a significant inverse correlation between levels of TARPs and levels …
Figure 4
Averaged ratios of GluN2B, NLG1, and NLG-2 to PSD-95 in mice and rat syngap1 HETs.
PSDs were purified as in Figure 2. Ratios were determined as described under Materials and methods. Ratios from HET animals (bars) are normalized to the ratios from WT animals (dotted lines). (A) …
Figure 5
Correlation of the ratios GluN2B/PSD-95 and synGAP/PSD-95 in individual animals.
Each point represents a single animal. Black, WT; Orange, HET. (A) All animals including all genotypes, ages, species, and sexes. n = 158. (B) All female animals, including all genotypes, ages, and …
Figure 6
Correlation of the ratios of NLG-2/PSD-95 or NLG-1/PSD-95 and synGAP/PSD-95 for individual animals.
Each point represents a single animal. Black, WT; Orange, HET. (A) Correlation of NLG-2/PSD-95. All animals, including all genotypes, ages, species, and sexes. n = 153. (B) Correlation of …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | Syngap1 KO mouse | Vazquez et al., 2004 | C57BL/6 | |
| Strain, strain background (Rattus norvegicus) | Syngap1 KO rat; LE-Syngap1em1/PWC | SAGE Labs, Sigma-Aldrich | Syngap1em1/PWC | CRISPR/Cas9 based genome targeting exon 8 |
| Antibody | anti-PSD-95 (Mouse monoclonal) | Caltech Monoclonal Antibody Facility | 50% ammonium sulfate cut of Ascites fluid of 7E3-1B8; AB_212825 | WB (1:10,000) |
| Antibody | anti-synGAP (Rabbit polyclonal) | Pierce | PA1-046; AB_2287112 | WB (1:3,500) |
| Antibody | anti-TARP (Rabbit polyclonal) | EDM Millipore | Ab9876; AB_877307 | WB (1:300) |
| Antibody | anti-GluN2B (Rabbit polyclonal) | Raised in our lab. Zhou et al., 2007, see Figure 2—figure supplement 1 | WB (1:1000) | |
| Antibody | anti-NLG-1 (Rabbit polyclonal) | Synaptic Systems | 129013; AB_2151646 | WB (1:2000) |
| Antibody | anti-NLG-2 (Rabbit polyclonal) | Synaptic Systems | 129202; AB_2151646 | WB (1:1000) |
| Antibody | Alexa Fluor 680 goat anti-mouse IgG | Thermo Fisher Scientific | A28183; AB_2536167 | WB (1 μg/ml) |
| Antibody | IRdye800-conjugated goat-anti-rabbit IgG | Rockland Immunochemicals, Limerick, PA | 611-145-122; AB_1057618 | WB (1 μg/ml) |
| Commercial assay or kit | BCA Protein Assay Kit | Pierce | 23227 | |
| Software, algorithm | Image Studio Light | LI-COR Biosciences | ||
| Software, algorithm | Microsoft Excel | Microsoft | ||
| Software, algorithm | Prism 8 | GraphPad Software, San Diego |
Additional files
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Supplementary file 1
Example calculations of synGAP/PSD-95 ratio and TARPs/PSD-95 ratio for animals 33 and 34.
The Table shows the steps to calculate a synGAP/PSD-95 ratio and a TARPs/PSD-95 ratio from digital data recorded from one pair of gels containing samples from animals 33 and 34 (see Figure 2—figure supplement 1). Data were collected from at least three technical replicates of synGAP gel lanes for each animal and usually six technical replicates of target gel lanes. The resulting ratios were averaged.
- https://cdn.elifesciences.org/articles/52656/elife-52656-supp1-v2.pdf
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Transparent reporting form
- https://cdn.elifesciences.org/articles/52656/elife-52656-transrepform-v2.pdf
