Supplementary Figure 2: Characterization of GluA1^C2KI and GluA2^C1KI mice

(a) Western blot analysis of total brain protein lysate from GluA1^C2KI and WT control littermates showing the successful replacement of the CTD of GluA1 with that of GluA2. Note in GluA1^C2KI mice, no GluA1 band was detected with a GluA1-CTD antibody, but a normal GluA1 band was seen with a GluA1-NTD antibody. (b) Western blot analysis of total brain protein lysate from GluA2^C1KI and WT control mice showing the successful replacement of the CTD of GluA2 with that GluA1. Note in GluA2^C1KI mice, no GluA2 band was detected with a GluA2-CTD antibody, but normal GluA2 level with a GluA2-NTD antibody. (c) Quantifications of various proteins tested in (a,b) showing that phosphorylated GluA1 (Ser831 and Ser 845) was absent in GluA1^C2KI, but increased in GluA2^C1KI compared to WT mice, and that phosphorylated GluA2 (Tyr869/873/876) was absent in GluA2^C1KI, but increased in GluA1^C2KI compared to WT mice. Other proteins were not altered except that there was a small increase in GluA1NTD in GluA2^C1KI mice (A1-NTD: WT = 1.00 ± 0.00, n = 4 mice; A1^C2KI = 0.86 ± 0.07, n = 4 mice, p = 0.085; GluA2^C1KI = 1.20 ± 0.05, n = 4 mice, *p = 0.023; F_{(2, 9)} =10.993, p = 0.004. A2-NTD: WT = 1.00 ± 0.00, n = 4 mice; A1^C2KI = 0.88 ± 0.04, n =4 mice; GluA2^C1KI = 1.16 ± 0.18, n = 4 mice; F_{(2, 9)} =1.743, p = 0.229. p-GluA1-831: WT = 1.00 ± 0.00, n = 4 mice; A1^C2KI = 0.08 ± 0.02, n = 3 mice, ***p = 0.00000002; GluA2^C1KI = 4.00 ± 0.89, n = 4 mice, *p = 0.015; F_{(2, 8)} =12.786, p = 0.003. p-GluA1-845: WT = 1.00 ± 0.00, n = 5 mice; A1^C2KI = 0.10 ± 0.02, n = 4 mice, ***p = 0.0000006; GluA2^C1KI = 4.84 ± 0.92, n = 5 mice, **p = 0.003; F_{(2, 11)} =19.077, p = 0.000265. p-GluA2-Tyr: WT = 1.00 ± 0.00, n = 4 mice; A1^C2KI = 1.51 ± 0.18, n = 4 mice, **p = 0.007; GluA2^C1KI = 0.15 ± 0.03, n = 4 mice, ***p = 0.000268; F_{(2, 9)} = 43.860, p = 0.000022. GluA3: WT = 1.00 ± 0.00, n = 4 mice; A1^C2KI = 1.08 ± 0.26, n = 4 mice; GluA2^C1KI = 1.09 ± 0.08, n = 3 mice; F_{(2, 8)} = 0.085, p = 0.919. GluA4: WT = 1.00 ± 0.00, n = 5 mice; A1^C2KI = 0.93 ± 0.11, n = 5 mice; GluA2^C1KI = 1.00 ± 0.10, n = 5 mice; F_{(2, 12)} = 0.621, p = 0.555. NR1: WT = 1.00 ± 0.00, n = 4 mice; A1^C2KI = 1.34 ± 0.09, n = 4 mice; GluA2^C1KI = 0.85 ± 0.19, n = 4 mice; F_{(2, 9)} = 4.180, p = 0.052. Syn1: WT = 1.00 ± 0.00, n = 4 mice; A1^C2KI = 1.04 ± 0.14, n = 4 mice; GluA2^C1KI = 1.10 ± 0.19, n = 4 mice; F_{(2, 9)} = 0.145, p = 0.867. PSD95: WT = 1.00 ± 0.00, n = 4 mice; A1^C2KI = 0.69 ± 0.06, n = 4 mice; GluA2^C1KI = 1.14 ± 0.19, n = 4 mice; F_{(2, 9)} = 3.945, p = 0.059). (d) Whole brain protein lysates immunoprecipitated with an anti-GluA1NTD antibody and probed with anti-GluA1NTD and anti-SAP97 antibodies showing reduced or absence of SAP97 in the immunocomplex in GluA1^C2KI mice. (e) Whole brain protein lysates immunoprecipitated with an anti-GluA2NTD antibody and probed with anti-GluA2NTD and anti-NSF antibodies showing reduced or absence of NSF in the immunocomplex in GluA2^C1KI mice. (f,g) Fixed brain sections immunostained with anti-synapsin 1 (green) and the nuclear marker DAPI (blue) showing normal hippocampal anatomy and synapse distribution in GluA1^C2KI (f) and GluA2^C1KI (g) mice compared to WT littermates. (h,i) Fixed brain sections immunostained with anti-GluA2-NTD (green) and the nuclear marker DAPI (blue) showing normal distribution of AMPARs in the hippocampus of GluA1^C2KI (h) and GluA2^C1KI (i) mice compared to WT littermates. Scale bar: 200 μm. Experiments were repeated at least 3 times for a,b,d and i. One-way ANOVA test was used for Fig. S2c followed by post-hoc Fisher’s LSD multiple comparison test for A1-NTD and p-GluA2-Tyr analysis, and by two tailed t-test at 95% confidence interval for p-GluA1-831 and p-GluA1-845 analysis. Data were presented as mean ± s.e.m. *p<0.05, **p<0.01, ***p<0.001. See Supplementary Fig. S10 for full length Western blot scans.