Odyssey imaging software

Human HTT protein levels in vitro were measured by western blot. Cells were pelleted and lysed in MSD Tris Lysis Buffer (Cat. No. R60TX; Meso Scale Discovery, Rockville, MD) supplemented with protease and phosphatase inhibitor cocktail. Cell lysates were then cleared by centrifugation and supernatants evaluated for total protein by the BCA assay. A sample containing 20 μg protein was denatured at 70°C and run on a 3–8% Tris Acetate NuPAGE^™ gel. Proteins were transferred onto PVDF membrane using the iBlot^™ 2 transfer device (Invitrogen) according to the manufacturer's instructions. The PVDF membrane was treated with LI-COR Odyssey^® blocking buffer and then incubated with primary antibodies against β-actin (Cat. No. ab8227; Abcam, Cambridge, MA) or HTT protein (Cat. No. MAB2166; EMD Millipore, Danvers, MA), followed by secondary antibodies comprising goat anti-mouse IRDye 800CW conjugated secondary antibody (LI-COR; 800 nm, green) for HTT protein and rabbit goat anti-rabbit IRDye 680RD conjugated secondary antibody (LI-COR; 680 nm, red) for β-actin. After extensive washing, the PVDF membrane was scanned with the LI-COR Odyssey imaging system at 680 and 800 nm, and the fluorescence intensities for the HTT and β-actin protein bands were quantified using the LI-COR Odyssey imaging software. For each sample, the HTT protein signal was normalized to the corresponding β-actin signal.

Cells were treated with 5 μM CC-115 with or without 1.5 mU/mL bleomycin for the indicated time and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.25% deoxycholic acid, 1% Nonidet P-40, 1 mM EDTA, and protease inhibitors). The cell lysates were subjected to SDS-PAGE and immunoblotting with indicated antibodies. NCI-H441 human lung cancer cells were treated with indicated concentrations of CC-115 with or without 1.5 mU/mL bleomycin for 2 hours and pDNA-PK in the cell lysates was measured using immunoblotting. The pDNA-PK bands were quantified by Odyssey imaging software (LI-COR, Lincoln, NE) and normalized to total DNA-PK. IC₅₀ values were determined by Excelfit (London, UK).

The enzymatic activities of tissue matrix metalloproteinases were measured by performing gelatin zymography in brain samples. Protein was extracted from fresh brain tissue in PBS, specifically the right posterior cerebral cortex and midbrain together, and quantified immediately using a BCA protein assay kit (Pierce Biotechnology) according to the manufacturer’s instructions. Protein samples were immediately separated on a precast 10% gelatin zymogram gel (Life Technologies). The gel was removed, incubated in zymogram renaturing buffer for 30 minutes, equilibrated for 30 minutes in zymogram developing buffer at room temperature and then incubated overnight at 37°C with gentle agitation in fresh zymogram developing buffer (all buffers obtained from Life Technologies). The next day, the gel was washed gently with water and incubated in IRDye blue protein stain, a Coomassie blue stain (LI-COR Biosciences, Lincoln, NE, USA). The gel was stained for 1 hour and then destained in H₂O until clear band resolution was apparent (approximately 1 hour). The gel was scanned on an Odyssey imager, and semiquantitative densitometric analysis was performed using Odyssey imaging software (LI-COR Biosciences).