Chloroquine

HeLa and HEK-293 T cells were purchased from ATCC and cultured in DMEM supplemented with 10% FBS and 5% Penicillin-Streptomycin, under conventional conditions. The day before transfection, cells were seeded in 12-well plates. Cells were transfected using Lipofectamine LTX (Invitrogen) per manufacturer’s directions. Twenty-four hours after transfection, cells were treated as indicated in figure legends with cycloheximide (100 μg/ml; A. G. Scientific), and/or MG132 (20 μM; A. G. Scientific) or chloroquine (100 μM, A. G. Scientific). Cells were harvested in hot SDS lysis buffer for western blotting (see below).

Ataxin-3 and eGFP variants were synthesized by the company Genscript (genscript.com) and subcloned into HA-tagged, pCDNA3.1(+1) for ataxin-3 or pCDNA3.1+N-eGFP for eGFP. The identity and integrity of all plasmids used in this work were confirmed through site-restricted digests as well as two rounds of sequencing by the vendor and by our group. HEK-293T cells were purchased from ATCC, confirmed for lack of mycoplasma contamination, and cultured in DMEM supplemented with 10% FBS and 5% penicillin-streptomycin, under conventional conditions at 37°C and 5% CO₂. The day before transfection, cells were trypsinized, resuspended, and seeded on 12-well or 24-well plates. Cells were then transfected with the plasmids indicated in figures using Lipofectamine LTX (Thermo Fisher) following the manufacturer’s protocols. The same total amount of plasmid was transfected into each well for each experimental group, helping to ensure consistent transfection efficiency. Twenty-four hours after transfection, cells were treated with MG132, chloroquine, or cycloheximide (each purchased from A. G. Scientific) as indicated in figures and legends.