0260 100 lc3 2g6

H4 neuroglioma cells were maintained, grown and transfected with HTT exon-1 fragments with a polyQ expansions of either 25 or 104 fused to GFP as in ref. 26 (link). Twenty-four hours after HTT25Q or HTT104Q transfection, cells in 35 mm imaging dishes (Ibidi), 60 mm or 100 mm dishes (Corning) were treated with vehicle (PBS) or MGO (0.5 mM) for 16 h. Total protein lysates were obtained as in ref. 13 (link). Htt was immunoprecipitated using GFP-trap (Chromotek) according to the manufacturer instructions. Widefield fluorescent microscope Zeiss Axiovert 200 M (Carl Zeiss MicroImaging) or point scanning confocal microscope Zeiss LSM 710 (Carl Zeiss MicroImaging) were used to visualize HTT inclusions. Immunobloting was performed according to standard procedures as in ref. 13 (link) using the following antibodies: anti GFP (NeuroMab, P42212, 1:3000); anti AGEs (Cosmobio, KAL-KH-001, 1:500); anti LC3 (Nano Tools, 0260-100/LC3-2G6, 1:2000) and anti β-actin (Ambion, AM4302, 1:5000). Cytotoxicity was measured by means of the LDH kit (Clontech), following manufacturer´s instructions. Cell viability was determined using the MTT assay according to standard procedures. Evaluation of HTT aggregation profiles by filter retardation assay was performed as in ref. 62 (link). Briefly, cellulose acetate membranes retain aggregates that are not soluble in SDS (1%) and that are larger than 0.22 μm.

Cells lysis and immunoblotting was performed as previously described [7 (link)]. For immunoblotting, we used the following antibodies: anti-aSyn (BD Transduction laboratories, S63320, 1:3000), anti-V5 (Santa Cruz, SC-83849-R, 1:1000), anti-LC3 (Nano Tools, 0260-100/LC3-2G6, 1:2000), anti–β-actin (Ambion, AM4302, 1:5000), anti-GAPDH (Ambion, AM4300, 1:5000), anti-GFP (NeuroMab, P42212, 1:3000), and anti–acetyl-lysine (Cell Signaling, 9441S, 1:1000).