Figure 2. 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 Materials and methods and in Figure 2—figure supplement 1. Ratios from HET animals (bars) are normalized to the ratios from WT animals (dotted lines). Antibodies against synGAP, TARPS, and PSD-95 are the same as those used in Walkup et al. (2016). The antibody against synGAP (AB_2287112) recognizes all isoforms of synGAP. The antibody against TARPs (AB_877307) recognizes TARP-γ2, γ3, γ4, and γ8. The sample sizes for each group and the significance tests are as follows. A) all animals WT = 79 and HET = 78, one-tailed Wilcoxon matched-pairs signed rank test; male mouse 7.5 weeks WT = 11 and HET = 9, one-tailed Student T-test; male mouse 12.5 weeks WT = 11 and HET = 8, one-tailed Student T-test with Welch’s correction; male rat 7.5 weeks WT = 11 and HET = 10, one-tailed Student T-test; male rat 12.5 weeks WT = 10 and HET = 11, one-tailed Student T-test; female mouse 7.5 weeks WT = 10 and HET = 12, one-tailed Student T-test with Welch’s correction; female mouse 12.5 WT = 9 and HET = 9, one-tailed Student T-test; female rat 7.5 weeks WT = 10 and HET = 10, one-tailed Student T-test; female rat 12.5 weeks WT = 9 and HET = 10, one-tailed Student T-test. B) all animals WT = 77 and HET = 80, two-tailed Wilcoxon matched-pairs signed rank test; male mouse 7.5 weeks WT = 10 and HET = 9, two-tailed Student T-test; male mouse 12.5 weeks WT = 10 and HET = 10, two-tailed Mann Whitney test; male rat 7.5 weeks WT = 10 and HET = 10, two-tailed Student T-test; male rat 12.5 weeks WT = 10 and HET = 1, two-tailed Student T-test; female mouse 7.5 weeks WT = 9 and HET = 10, two-tailed Student T-test; female mouse 12.5 WT = 9 and HET = 10, two-tailed Mann Whitney test; female rat 7.5 weeks WT = 10 and HET = 10, two-tailed Student T-test; female rat 12.5 weeks WT = 9 and HET = 10, two-tailed Student T-test with Welch’s correction. Significance: * for p≤0.05, ** for p≤0.01, *** for p≤0.001, and **** for p≤0.0001.

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 described under Materials and methods. The numbers at the top of each lane identify samples from individual rodents. The fluorescent signals at 700 and 800 nm on double-stained blots were scanned separately on the LiCor Odyssey scanner and stored in separate files. The figure is a superposition of images of the two scans. The brightness of each image was adjusted to provide the most accurate visual appearance. The LiCor system has two important advantages; adjustment of the brightness and contrast of images does not alter the underlying digital data; and the digital data can be recorded over a very large linear range. The images show examples of ‘regions of interest (ROI)’ placed with LiCor software on each of the four underlying digital files. Digital fluorescence densities within each ROI were recorded and transferred to Excel. To record background densities We used similarly sized boxes placed immediately below the target fluorescent bands. The bkg. boxes are shown only in B) to avoid visual confusion, but were recorded for all signals. (C) Specificity of antibodies for neuroligin-1 and GluN2B. For staining of synGAP, TARPS, and neuroligin-2, we used the same antibodies that were documented in Walkup et al. (2016). However, we used a different antibody to label NLG-1 (rabbit anti-NLG-1 (Synaptic Systems 129013 AB_2151646) and to label GluN2B we used rabbit anti-GluN2B). The figure shows blots of PSD fractions souble-labeled with anti-PSD-95 and these last two antibodies, to document their specificity.