Proteinase inhibitors cocktail complete

For polyQ protein quantification, treated young adult animals were washed with M9 1X buffer before lysing the samples with RIPA buffer (Invitrogen, Carlsbad, CA, USA) and proteinase inhibitors cocktail (Complete, Roche, Basel, Switzerland). Samples were boiled at 100 °C for 10 min containing 4X SDS sample loading buffer. Protein extracts were separated by 8% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene di-fluoride (PVDF) membranes (Bio-Rad Laboratories, Hercules, CA, USA) by semi-dry blotting (Trans-Blot Turbo, Bio-Rad). Blocking was done with 3% milk, according to the specification of the following primary antibodies: mouse anti-polyQ (1:1000, Sigma Ref. #P1874) and mouse anti-actin (1:500, Invitrogen ref. #MA5–11869) to normalize values. Primary antibodies were incubated overnight at 4 °C degrees with shaking. We used the secondary antibody anti-mouse conjugated to HRP (1:10000, Santa Cruz ref. #SC-2005, Santa Cruz Biotechnology, Dallas, TX, USA) to develop immunoblots. Images were obtained using Amersham Imager 600 and enhanced chemiluminescent (ECL) detection (GE Healthcare, Chicago, IL, USA). Quantification values were obtained using the Image J software.

Protein was extracted from SCC cell lines using RIPA Lysis buffer, (Millipore, Temecula, CA). PhosSTOP and proteinase inhibitors cocktail (complete) (Roche Diagnostic GmbH, Mannheim, Germany) were added according to manufacturer’s instructions. Proteins were resolved using NuPAGE 4–12% Bis-Tris Gel (Invitrogen, Carlsbad, CA), transferred to PVDF membrane (Bio-Rad, Hercules, CA), and probed with primary antibodies (Table S1).