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Figure S1:Expression of GAPDH is not significantly altered by genotype or DHPG treatment. A two-way ANOVA indicated no significant main effect of genotype (F1,8 = 2.55, P > 0.05), treatment (F1,8 = 3.05, P > 0.05), or genotype × treatment interaction (F1,8 = 0.14, P > 0.05). Moreover, pairwise comparisons within each genotype failed to detect any significant differences in GAPDH levels after DHPG treatment (Fmr1WT: t4 = 1.22, P > 0.05; Fmr1KO: t4 = 1.38, P > 0.05; two-sample t test). For these reasons, we normalized all protein signals in Fig. 1 to GAPDH. Similar results were obtained when normalizing to ERK1/2 and tubulin (data not shown); however, we had concerns using either of these in this study. Specifically, ERK1/2 is a STEP substrate and phosphorylation of ERK1/2 is dysregulated in Fmr1KOmice (Paul et al. 2003; Kim et al. 2008; Osterweil et al. 2010). Moreover, because abnormal dendritic spine morphology is a hallmark of Fmr1KO mice and in FXS humans (Comery et al., 1997), we felt that use of tubulin for normalization was inappropriate because of its role in regulating cytoskeletal dynamics.

Figure S2:No significant differences are detected among genotypes in sociability or preference for social novelty in the social choice task. (a) Pairwise comparisons indicate that each genotype spends significantly more time in close proximity to the mouse compared with the cup [*Ps ≤ 0.05, two-sample t test: STEPWT/Fmr1WT (t14 = 2.41), STEPHT/Fmr1WT (t20 = 5.09), STEPKO/Fmr1WT (t10 = 5.43), STEPWT/Fmr1KO (t24 = 6.72), STEPHT/Fmr1KO (t14 = 5.68) and STEPKO/Fmr1KO (t10 = 5.35)], indicating appropriate sociability. (b) No significant genotypic differences are found in the socialization index (% of time spent in close proximity to the mouse relative to the total time spent in close proximity to the mouse and cup). (c) Pairwise comparisons indicate that none of the genotypes spend more time with the novel mouse than the familiar one [Ps > 0.05, two-sample t test: STEPWT/Fmr1WT (t14 = −0.31), STEPHT/Fmr1WT (t20 = 04), STEPKO/Fmr1WT (t10 = 0.73), STEPWT/Fmr1KO (t24 = 1.96), STEPHT/Fmr1KO (t14 = 1.18) and STEPKO/Fmr1KO (t10 = 0.23)], suggesting lack of social novelty preference. (d) No significant genotypic differences are found in the social novelty index (% of time spent in close proximity to the novel mouse relative to the total time spent in close proximity to the novel and familiar mice). Data represent the mean for each measure ± SEM.

Figure S3:No significant differences are detected among genotypes in open-field activity or marble burying. (a) Total distance traveled in the open-field arena (5 min) is not significantly different between groups. (b) Genotype also does not affect the ratio of center to total distance traveled in our study. (c) The number of marbles buried 40% does not differ significantly between genotypes. Data represent the mean for each measure ± SEM [open-field activity: STEPWT/Fmr1WT (n = 12), STEPHT/Fmr1WT (n = 14), STEPKO/Fmr1WT (n = 9), STEPWT/Fmr1KO (n = 20), STEPHT/Fmr1KO (n = 17) and STEPKO/Fmr1KO (n = 7); marble burying: STEPWT/Fmr1WT (n = 11), STEPHT/Fmr1WT (n = 14), STEPKO/Fmr1WT (n = 11), STEPWT/Fmr1KO (n = 17), STEPHT/Fmr1KO (n = 11) and STEPKO/Fmr1KO (n = 10)].

Table S1:Original numbers of mice included in each cohort for behavioral analysis. A total of eight mice (three STEPWT/Fmr1KO and five STEPHT/Fmr1KO) were assessed in open field but did not complete the remainder of the behavioral battery because they died from natural causes before the experiment. (SZ, seizure, n.e., not examined).

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