Figure 6. Mutant Htt affects Ku70 and... | Rockefeller University Press

Figure 6.

$Figure 6. Mutant Htt affects Ku70 and Ku80 expression in primary cortical neurons. (A) By Western blotting, Ku70 and Ku80 expression in primary cortical neurons was investigated chronologically after infection of AxCA, AxCAhtt20Q, (Htt20Q), or AxCAhtt111Q (Htt111Q). (B) Mutant Htt does not affect expression of DNA–PKcs. Immunoblot detection of the change of DNA–PKcs expression in cultured neurons at 2 and 4 d after virus infection. Cultured neurons infected by AxCA, AxCAhtt20Q, (Htt20Q), or AxCAhtt111Q (Htt111Q) were collected, and the cell lysates (5 µg) were immunoblotted by anti-DNA–PKcs (H-163) antibodies. (C) The chronological change of Ku70 expression in cytosol and nucleus in cultured neurons. 1 µg nuclear (N) or cytosol (C) protein extract was blotted by anti-Ku70 antibody (H-308). Detection of GAPDH and HDAC3 was used to determine the purity of the fractions. Ku70 expression changed only in the nucleus. The lower GAPDH band in the nuclear fraction might be the nuclear translocation form (Sawa et al., 1997; Dastoor and Dreyer, 2001). (D) Effect of proteasome inhibitor on Ku70 protein levels in neurons was examined by immunoblots. Neurons infected by AxCA, AxCAhtt20Q (Htt20Q), or AxCAhtt111Q (Htt111Q) cultured in the presence or absence of proteasome inhibitor MG-132 were used for the analysis. 0.3 µM MG-132 was added to the culture medium 2 d after virus infection (day 2). Neurons were collected after 2 d of treatment with or without MG-132 (day 4).$

Mutant Htt affects Ku70 and Ku80 expression in primary cortical neurons. (A) By Western blotting, Ku70 and Ku80 expression in primary cortical neurons was investigated chronologically after infection of AxCA, AxCAhtt20Q, (Htt20Q), or AxCAhtt111Q (Htt111Q). (B) Mutant Htt does not affect expression of DNA–PKcs. Immunoblot detection of the change of DNA–PKcs expression in cultured neurons at 2 and 4 d after virus infection. Cultured neurons infected by AxCA, AxCAhtt20Q, (Htt20Q), or AxCAhtt111Q (Htt111Q) were collected, and the cell lysates (5 µg) were immunoblotted by anti-DNA–PKcs (H-163) antibodies. (C) The chronological change of Ku70 expression in cytosol and nucleus in cultured neurons. 1 µg nuclear (N) or cytosol (C) protein extract was blotted by anti-Ku70 antibody (H-308). Detection of GAPDH and HDAC3 was used to determine the purity of the fractions. Ku70 expression changed only in the nucleus. The lower GAPDH band in the nuclear fraction might be the nuclear translocation form (Sawa et al., 1997; Dastoor and Dreyer, 2001). (D) Effect of proteasome inhibitor on Ku70 protein levels in neurons was examined by immunoblots. Neurons infected by AxCA, AxCAhtt20Q (Htt20Q), or AxCAhtt111Q (Htt111Q) cultured in the presence or absence of proteasome inhibitor MG-132 were used for the analysis. 0.3 µM MG-132 was added to the culture medium 2 d after virus infection (day 2). Neurons were collected after 2 d of treatment with or without MG-132 (day 4).