Western blot analysis and ?lter retardation assay The cells were transfected as described above. After 48 h from transfection, cells were harvested and centrifuged 5 min at 1.2 rpm at 4 C; the pellets of cells were resuspended in PBS (added of the protease inhibitors cocktail, Sigma-Aldrich) and homogenized using slight sonication as previously described ( Poletti et al., 2001 ). Total proteins were determined with the bicinchoninic acid method (BCA assay, Pierce, IL, USA). Western immunoblot analysis was performed on 12% SDS polyacrylamide gel electrophoresis () loading 30  g of total proteins. Samples were then electro-transferred to nitrocel- lulose membranes (Trans-blot, Bio-Rad Mycophenolate mofetil Laboratories, CA, USA) or PVDF (for detecting LC3, Polyscreen transfer membrane, PerkinElmer, MA, USA) using a liquid transfer apparatus (Bio-Rad). The mem- branes were treated with a blocking solution containing 5% non-fat dry milk in Tween-TBS (TBS-T, 20 mM TrisHCl, pH 7.5, 0.5 M NaCl, 0.05% Tween-20) for 1 h and then incubated with the primary 2 P. Rusmini et al. / Neurobiology of Disease 41 (2011) 83 ?95 85 antibodies:

(a) rabbit polyclonal ARN-20 (sc-816, Santa Cruz; dilution 1:500) to detect wt AR and ARpolyQ (b) rabbit polyclonal anti-Cu/Zn superoxide dismutase SOD1 (SOD-100; Assay Designs, MI, USA; dilution 1:1000) to detect the wt and G93A-SOD1 proteins; (c) mouse monoclonal anti-FLAG (Sigma-Aldrich) to detect FL or  C TDP-43; (d) rabbit polyclonal anti-LC3 (L8918, Sigma-Aldrich; dilution 1:1000) to detect the autophagosomal marker LC3, a protein essential for autophagosome; (e) mouse monoclonal anti- Hsp90 (SPA-830, Assay Designs; dilution 1:1000) to detect Hsp90 level; (f) rabbit polyclonal anti-Hsc70/Hsp70 (SPA-757, Assay Designs; dilution 1:1000) to detect Hsp70 level; (g) goat polyclonal anti-Actin (Actin I-19; Santa Cruz, dilution 1:1000) to detect total actin; (h) peroxidase labeled anti-GFP (Vector Laboratories, CA, USA; dilution 1:5000) to detect Mycophenolate mofetil 128794-94-5 YFPu. Immunoreactivity was detected using the following sec- ondary peroxidase-conjugated antibodies: goat anti-rabbit (sc-2004, Santa Cruz, dilution 1:5000) was used to identify both the anti-AR, the anti-SOD1, the anti-LC3 and the anti Hsp70; goat anti-mouse (sc-2005, Santa Cruz, dilution 1:5000) was used to identify the anti- FLAG and the anti-Hsp90;

donkey anti-goat (sc-2020, Santa Cruz, dilution 1:5000) was used to identify the anti-Actin antibody. The immunoreactive regions were then visualized using the enhanced chemiluminescence detection kit reagents (ECL plus, GE Healthcare, UK). The same buy Mycophenolate mofetil membranes were subsequently processed with different antibodies to detect the levels of different proteins in the same samples loaded on the gel, after stripping for 25 min at 37 C in Restore Western blot stripping buffer (Pierce). Filter retardation assay was performed by sample ?ltration through a 0.2-  m cellulose acetate membrane (Whatman, Germany) using a slot-blot apparatus (Bio-Rad) and loading 1.5  g of total proteins for AR.Q(n) and TDP-43 s samples; 0.75  g were used in the case of SOD1s analysis. Slot-blots were probed as described for western blots.

Optical intensity of samples assayed with ?lter retardation assay or western blot was detected and analyzed using NIH ImageJ software. Cyto ?uorimetric analysis The cells were transfected as described above. The transfected cells were harvested primal cut  and centrifuged 5 min at 1200 rpm at 4 C; the cell pellets were resuspended in 400  l of 4% paraformaldehyde, incubat- ed at RT for 10 min on a rotator and then centrifuged 5 min at 1200 rpm at 4 C. Once supernatant was aspirated from cell pre- paration, the pellets were resuspended in 300  l of 4% FBS PBS. Cell ?uorescence was detected using FACS Calibur (BD Pharmingen). Flow cytometry results were analyzed using CellQuest (BD Pharmingen) program analysis software. Cell viability assay The 3-(4,5-dimethyl-2-thiazolyl)-2,5 diphenyl-2H-tetrazolium bromide (MTT)-based cell proliferati