Journal: The Journal of Cell Biology
Article Title: Mutant huntingtin impairs Ku70-mediated DNA repair
Figure Lengend Snippet: Protein–protein interaction between Ku70 and mutant Htt. (A) IP assay with mouse brain samples. The nuclear extract from brain samples (cerebral cortex and striatum) of R6/2 or nontransgenic littermate (wt) mice at 9 wk were precipitated with anti-Ku70, -1C2, or -HD1 antibody and blotted with the other antibody. In the case of HD1 antibody, wild-type full-length Htt and aggregation of mutant Htt at the gel top are shown in input (arrow). In output, only the mutant Htt was coprecipitated with Ku70. As negative controls, mouse and rabbit IgG were used for precipitation. (B) IP assay showing interaction between endogenous Ku70 and overexpressed mutant Htt88Q exon 1 in HeLa cells. Interaction between normal Htt and endogenous Ku70 was not clear in this condition. (C) Deletion analysis demonstrated that mutant Htt interacts with the N-terminal domain of Ku70. (top) Diagram shows the constructs for the deletion analysis. (bottom) Coprecipitation pattern of GFP-HttQ88 with various Flag-Ku70 deletion mutants. NLS, nuclear localization signal. (D) The amount of mutant Htt necessary for interaction with endogenous Ku70 was titrated with Tet-on stable cells in which mutant Htt expression can be regulated by the concentration of tetracyclin in the medium. (top) Lanes 1–5 indicate a semiquantitative RT-PCR experiment with Htt-specific primers using cDNAs obtained from the Tet-on EGFP-HttQ88 stable cells treated with tetracyclin at different concentrations (0–100,00 pg/ml) for 48 h. The Htt-specific primers are designed to amplify the CAG repeat region to distinguish the endogenous Htt and induced EGFP-HttQ88 genes. The single asterisk denotes induced GFP-HttQ88 transcripts, the double asterisk denotes endogenous Htt transcripts (lanes 1–5). As a control, corresponding PCR with GAPDH-specific primers was performed. As a semiquantitative PCR calibration, PCR was performed with the Htt primers using plasmid DNAs, pEGFP-HttQ17 (lanes 6–11), and pEGFP-HttQ88 (lanes 12–17) as PCR templates with indicated amounts to evaluate the RT-PCR. (middle) Equal amounts of cell lysate from the Tet-on stable cell lines treated with the indicated concentration of tetracyclin for 48 h were blotted with anti-GFP antibody. (bottom) The lysates were also subjected to IP with anti-GFP antibody, and the precipitants were blotted with anti-Ku70 and GFP antibodies. IB, immunoblot.
Article Snippet: The dilution conditions of primary and secondary antibodies for immunoblotting were as follows: rabbit anti-CAG53b (1:1,000; ), mouse anti-GFP (1:2,000; Takara Bio Inc.), rabbit anti-GST (1:500; Z-5; Santa Cruz Biotechnology, Inc.), mouse anti-polyQ (1:2,000; 1C2; Millipore), mouse anti-Ku70 (1:500; N3H10; Kamiya Biomedical), rabbit anti-Ku70 (1:1,000; H-308; Santa Cruz Biotechnology, Inc.), rabbit anti-Ku80 (1:1,000; H-300; Santa Cruz Biotechnology, Inc.), rabbit anti-DNA–PKcs (1:500; H-163; Santa Cruz Biotechnology, Inc.), mouse anti-γH2AX (1:500; Ser139; Millipore), rabbit anti-HD1 (1:2,000; ), rabbit anti-HDAC3 (1:500; Millipore), mouse anti-GAPDH (1:10,000; Millipore), HRP-conjugated anti–goat IgG (Dako), anti–mouse IgG (GE Healthcare), and anti–rabbit IgG (1: 3,000; GE Healthcare).
Techniques: Mutagenesis, Mouse Assay, Construct, Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Plasmid Preparation, Stable Transfection