FIGURE 1.
In the mouse neocortex and hippocampus, FMRP loss leads to altered levels of select scaffold proteins and receptor subunits in the PSD. A, proteins (15 μg per lane) from soluble protein (lane 1), crude membrane P2 (lane 2), synaptosomal (lane 3), and PSD fractions (lane 4) of adult wild-type mice were analyzed via Western blotting with antibodies directed against NR1 and β-tubulin. Levels of the PSD marker protein NR1 increase in consecutive fractions obtained during PSD purification. The upper panel represents a shorter exposure of the blot shown in the middle panel. B, Western blot analysis of proteins from the soluble brain protein fraction (15 μg per lane) of 2-month-old wild-type (wt) and Fmr1−/− (ko) mice. Although β-tubulin is detected in both protein extracts, FMRP is only present in lysates obtained from wild-type animals. C, in Western blots performed with PSD fractions from the neocortex and hippocampus of 2-month-old wild-type and Fmr1−/− mice (7 μg per lane), different proteins (as indicated) were detected with specific antibodies. D, bar graph indicating the knock-out to wild-type ratio (ko/wt ratio) in the levels of individual proteins detected in Western blot experiments as shown in C. For each PSD component with an altered level in knock-out as compared with wild-type mice, the final value is based on at least three different PSD preparations and three or more independent Western blot experiments per preparation. For proteins with unchanged levels, at least two independently prepared PSD fractions were investigated and each by two or more immunoblotting experiments. Protein levels were determined via densitometric evaluation of the immunochemical signal and normalized to the signal intensity obtained for β-tubulin. A bar value of 1 indicates that the normalized levels of the respective protein in PSD preparations from knock-out and wild-type animals are identical. Simple horizontal lines specify standard deviations. Altered knock-out to wild-type ratios as observed for SAP97 (neocortex, p = 0.001, linear mixed models application), Chapsyn-110 (neocortex, p = 0.031), SAPAP1 (neocortex, p = 0.001), SAPAP2 (hippocampus, p < 0.001), SAPAP3 (hippocampus, p < 0.001), Shank1 (neocortex, p < 0.001; hippocampus, p < 0.001), Shank3 (neocortex, p < 0.04), IRSp53 (hippocampus, p = 0.005), NR1 (neocortex, p = 0.013; hippocampus, p < 0.001), NR2B (hippocampus, p = 0.016), and GluR1 (neocortex, p = 0.001) are statistically significant (marked by asterisk). E, Western blots were performed with PSD fractions from the neocortex and hippocampus of 2-week-old wild-type (wt) and Fmr1−/− (ko) mice (7 μg per lane) and antibodies directed against different postsynaptic components (as indicated). F, bar graph indicating the knock-out to wild-type ratios of the postsynaptic protein levels determined in Western blot experiments as shown in E. Altered knock-out to wild-type ratios as observed for SAPAP1 (neocortex, p = 0.01, linear mixed models application), SAPAP2 (hippocampus, p = 0.028), SAPAP3 (neocortex, p = 0.03), Shank1 (neocortex, p = 0.012), NR1 (neocortex, p < 0.001; hippocampus, p = 0.008), and GluR1 (neocortex, p = 0.024) are statistically significant (marked by asterisk). For further details see text.