Loading buffer 4x

Expression of the target protein was evaluated by analyzing a sample of the supernatant from 10 recombinant clones of Lt-RBD by Western blotting. After 72 h of growth in BHI complete medium, supplemented with NTC at 26°C, Leishmania cultures were centrifuged 10 min at 3,000 g. Clarified supernatants were filtered using a 0.22-μm nitrocellulose membrane and concentrated at 5,000 g for 30 min, using an Amicon ultracentrifugal filter with a molecular weight (MW) cutoff of 10 kDa. Samples were diluted in a loading buffer 4X (Thermo Fisher Scientific), boiled for 5 min, and subsequently loaded onto a 4–20% gradient polyacrylamide gel (Bio-Rad Laboratories). After electrophoresis, proteins were transferred onto a nitrocellulose membrane (Bio-Rad Laboratories), according to the standard protocols, before blocking for 5 min at room temperature with EveryBlot Blocking Buffer (Bio-Rad Laboratories) and incubation with a 1:3,000 dilution of anti–6xHis-tag–horseradish peroxidase (HRP) antibody (Thermo Fisher Scientific) in the EveryBlot Blocking Buffer for 1 h. After three washes with phosphate-buffered saline (PBS) + 0.1% (v/v) Tween 20 (PBS-T), the membrane was incubated for 5 min with the Clarity Western ECL Substrate (Bio-Rad Laboratories) and detected using the ChemiDoc Touch Imaging System (Bio-Rad Laboratories).

Cells overexpressing UHRF1 were treated over-night with MG-132 at 5 μM as final concentration. Next, cells were lysed with RIPA buffer supplemented with MG-132 1 μM, DUB inhibitor PR-619 and beta-Mercaptoethanol. 500 μg of cell lysates immunoprecipitated using UHRF1 antibody (Santa Cruz) following manufacturer’s protocol. Protein A/G coated beads were added to the mix, and everything was incubated over-night a 4 C in rotation. The next day, beads were washed five times with lysis buffer and resuspended with 50 μl Loading buffer 4X (Thermo Fischer) plus Reducing Agent 1X (Thermo Fischer). Western blotting was then performed, and samples were immunoblot with Ubiquitin antibody. The amount of UHRF1 immunoprecipitated was analysed using 5 μl of immunoprecipitated lysates, while inputs were loaded and immunoblotted a part in order to control quality of equal loading of different samples (using Tubulin as housekeeping).