Detailed Western Blot and Immunofluorescence Protocols for Huntingtin Protein

For western blot: polyclonal anti-Huntingtin 1–17 Ab1(1μg/ml) [39 (link)], α-actinin-2 (1:2000; ab68167; Abcam; specific [37 (link)], α-actinin (2μg/ml; ab18061; Abcam; may cross react with isoforms 1–4), GAPDH (1:6000, MAB374, Millipore). For fluorescent labeling: Anti-Huntingtin Ab2527 and Ab1173 were previously described [40 (link), 41 (link)]. Both are rabbit polyclonal made against peptides covering aa 2527–2547 and aa 1173–1196 of Huntingtin, respectively and were used at 3 μg/ml. The immunogen peptides consisted of the following amino acids: “ySCLEQQPRNKPLKALDTRFGR” and “ySLTNPPSLSPIRRKGKEKEPGEQA”. Anti-α-actinin (1:500; ab18061; Abcam; may cross react with isoforms 1–4), vinculin (1:100; Sigma). Secondary antibodies include Cy3 goat anti-mouse secondary antibody (1:500; Jackson Immunoresearch); Cy3 goat anti-rabbit secondary antibody (1:500; Jackson Immunoresearch); Bodipy Green goat anti-mouse secondary antibody (1:500; Invitrogen). Stains include Alexa Fluor 488-Phalloidin and Rhodamine-Phalloidin (1:500; Molecular Probes) for F-actin, and DAPI (1:500; Sigma) or Hoechst stain (1:1000; Molecular Probes) for nuclei. For peptide blocking experiments, 3 μg/ml Ab2527 in 4% Normal Goat Serum/PBS was incubated overnight with 30 μg/ml peptide (above) overnight at 4°C or with an unrelated peptide (yEPGDQENKPCRIKGDIGQST).