Figure 4.

Optimization of tamoxifen treatment protocol in adult Shank3^GCre^Tam+ mice. A, Quantification of Western blotting showing minimal rescue of WT SHANK3 protein expression following treatment with 4-hydroxytamoxifen (66.67 mg/kg) given once per day subcutaneously for 15 d (n = 7 for all treatment groups). B, Representative Western blotting (top) and quantification of whole-brain lysates (bottom) showing degrees of rescue of SHANK3 protein expression after varying duration of tamoxifen diet (n = 7 for all treatment groups). C, SHANK3 protein levels from whole-brain lysates in adult Shank3^GCre^Tam− mice treated for six weeks with vehicle or tamoxifen diet (vehicle diet: Shank3^+/+ n = 9, Shank3^+/G n = 7, Shank3^G/G n = 4; tamoxifen diet: Shank3^+/+ n = 12, Shank3^+/G n = 9, Shank3^G/G n = 9). D, SHANK3 protein levels from whole-brain lysates in adult Shank3^GCre^Tam+ mice treated for six weeks with vehicle diet or tamoxifen diet [vehicle diet: Shank3^+/+ n = 26, Shank3^+/G n = 21, Shank3^G/G n = 15; tamoxifen diet: Shank3^+/+ n = 23, Shank3^+/G n = 15, Shank3^G/G n = 18; data are normalized to the β-actin control and then to the average of WT levels with C-terminal SHANK3 antibody (JH3025)]. E, Example Southern blotting of brain tissue in Cre^Tam+, WT and Shank3^G/G homozygous mutant mice treated with vehicle or tamoxifen (TAM; Rev = band expected following cre-mediated recombination of floxed mutant; Mut = band expected for floxed mutant before cre recombination; WT = band expected in WT mice without mutant allele); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Error bars represent S.E.M. in this and all subsequent figures.