Figure 7.

Phosphorylation may reduce toxicity but increase insolubility of Htt. (A) Mimicking 97QP Httex1p phosphorylation reduces its toxicity in acutely transfected rat cortico-striatal slice explants. Plasmids encoding YFP and 97QP-CFP, 97QP DD-CFP, 97QP AA-CFP, or CFP control were biolistically cotransfected into rat cortico-striatal brain slices. The number of healthy medium spiny neurons in the striatal region of each slice was scored 5 d after transfection. n = 9 for each condition. Error bars represent SEM. Asterisk = difference from 97QP at P < 0.0001. (B) Immunoprecipitated phosphorylated/acetylated wt Htt purified from mouse brain runs in an insoluble fraction in the SDS-PAGE stacking gel. 12 independent wt control (W) or R6/2 (R) mouse brains were collected at 4, 8, and 12 wk of age, snap frozen, and lysed. 500 µg of lysate was subjected to immunoprecipitation with PW0595 anti-Htt antibody or zero antibody control (lysate from lanes 12 and 13 are identical). Western analysis was performed with a series of antibodies listed to the right of the panel. Immunoprecipitated Htt is present as an insoluble species in the stacking gel and at the top of the separating gel, and as a soluble form at the standard 350-kD size, marked with an arrow. MAB2166 does not detect the insoluble species well.