(A) Schematic of the recombineered BAC cassette, in which S421D or S421A mutations within the original BACHD construct were generated. (B) Representative Western blot demonstrating expression levels of mHTT from cortical lysates of 2-month-old BACHD, mHTT-S421D, and mHTT-S421A mice. The blot was probed with anti-expanded polyQ mAb 4H7H7 and anti–γ-tubulin as a loading control. (C) Quantification of expression levels of mHTT from cortical lysates of 2-month-old BACHD, mHTT-S421D, and mHTT-S421A mice. Values are based on the mean of 3 independent 4H7H7 blots, each with lysates from at least 3 mice per line compared across different blots by normalization to BACHD samples. Each value was first normalized for input using the anti–γ-tubulin control. F = 36.71, S421A vs. BACHD/S421D, P < 0.0001. (D) Quantification of the levels of cortical mHTT transcript in BACHD, mHTT-S421D, and mHTT-S421A mice by qRT-PCR. The results are from 4 independent samples per transgenic line, each run in quadruplicate. Values are normalized to BACHD mice. F = 51.8, P < 0.0001; S421A vs. BACHD/S421D, P < 0.0001. **P < 0.01; ***P < 0.001; ****P < 0.0001; all by 1-way ANOVA.