The Journal of Neuroscience, July 4, 2007, ():
Phosphorylation of Huntingtin by Cyclin-Dependent Kinase 5 Is Induced by DNA Damage and Regulates Wild-Type and Mutant Huntingtin Toxicity in Neurons
J. Neurosci. Anne et al.
27: 7318
Supplemental Data
Files in this Data Supplement:
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Supplemental material
- supplemental material
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Supplemental Figure 1. Huntingtin expression levels are unchanged in neurons upon serine in alanine and serine in aspartic acid mutations at S1181/1201. A, Primary cultures of striatal neurons are transfected by a modified phosphate calcium technique at 3 DIV or electroporated at 0 DIV with constructs encoding the first 1301 amino-acids of human huntingtin containing 17 (wild-type, 1301-17Q) or 73 glutamines (1301-73Q) with intact serines or serines to alanines or aspartic acids mutations. Huntigtin expression level is assessed 24 hr after transfection by immunoblotting with anti-htt 4C8. Anti-α-tubulin is used as loading control. B, Expression of the first 1301 amino-acids of human huntingtin in neurons is higher after transfection compared to electroporation but the efficiency of transfection is better using electroporation. White stars indicate transfected neurons. Neurons are fixed in 4% paraformaldehyde 24 hr after transfection and cells are immunostained with anti-htt 4C8 antibody. The nuclear staining is shown in blue. Scale bar: 100 μm.
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Supplemental Figure 2. Total and S1201-phosphorylated huntingtin and Cdk5/p35 are present in the cytosol and in the nucleus. Anti-α-tubulin and histone H3 antibodies are used as controls for proper cytosol/nuclear fractionation.
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Supplemental Figure 3. Constitutive phosphorylation of serines 1181 and 1201 blocks polyQ-huntingtin induced neuronal death. Primary cultures of striatal neurons are transfected by a modified phosphate calcium technique with 1301-73Q constructs with intact serines or serines to alanines or aspartic acids mutations. Striatal neurons are immunostained using anti-htt 4C8 antibody (red). Nuclei are stained with Hoechst. White arrows indicate degenerating neurons. Scale bar, 100 μm.
