Tube2 magnetic beads

HEK-293T cells were seeded in 10-cm dishes and transfected with FLAG-LRRK2 in the presence of myc-TRIM1 or myc-alone vector control. After transfection for 8 h, cells were treated with 100 nM bortezomib for 12 h. 20 h after transfection, cells were lysed as above, and lysate protein concentrations were normalized using BCA assay. FLAG-LRRK2 immunoprecipitation was performed using 30 μl FLAG antibody-conjugated beads (Sigma-Aldrich) at 4°C for 1 h. FLAG-LRRK2 was eluted by incubation with 100 µg/ml 3× FLAG peptide at 4°C for 1 h. 50 μl TUBE2 magnetic beads (LifeSensors) was washed 3× in TBS-T, added to eluted FLAG-LRRK2, and rotated at 4°C overnight. TUBE2 beads were washed 3× (50 mM Tris, pH 7.5, 250 mM NaCl, 0.2% NP-40, and 1 mM DTT), and bound proteins were eluted by heating at 70°C for 10 min in 20 μl 4× Nupage LDS loading buffer (NP0007; Invitrogen) containing 5% β-mercaptoethanol. Standards used were K48- and K63-linked recombinant polyubiquitin chains (R&D Biosystems).

Ubiquitination of endogenous proteins was determined using a TUBE pull-down assay. Briefly, cells were treated with MG132 (10 μM) for 12 h before lysis. Cells were lysed in TUBE lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 10% glycerol) supplemented with protease inhibitors (Roche), 1,10-phenanthroline (MCE, HY-W004544) (5 mM), N-Ethylmaleimide (Topscience, T3088) (5 mM) and PR-619 (MCE, HY-13814) (100 µM). Cell lysates were clarified by centrifugation and incubated with TUBE2 magnetic beads (LifeSensors, UM-0402M-1000) at 4 °C for 2 h. To enrich Flag-tagged TUBEs, clarified cell lysates were immunoprecipitated with anti-Flag® M2 Affinity Gel (Sigma‒Aldrich, A2220). Beads or gels were then washed three times and boiled with 4× SDS loading buffer. The precipitated proteins were subjected to western blot analysis with the indicated antibodies.