Sds page loading buffer

35 K-Fc was detected in plasma by immunoprecipitation with Protein A/G agarose beads (Sigma, UK). Plasma samples were diluted 1:2 with Cell-lytic MT lysis buffer (Sigma, UK) and incubated with Protein A/G beads overnight at 4 °C with rotational mixing. The beads were washed 5 times with the lysis buffer prior to solubilisation of the bound proteins by LDS containing SDS-PAGE loading buffer (Invitrogen, UK). The resulting protein fraction was run on a western blot on a Nu-Page gel (Invitrogen) and transferred onto PVDF membrane (Amersham). The proteins were detected by blotting with anti-35 K antibodies (R&D Systems) and anti-human Fc (Jackson Labs, US) antibodies.

USP1–UAF1 complexes were assembled and used to make 2× DUB stocks at 200 nM, or as indicated in Fig 3C. Ubiquitinated substrates were made as 2× stocks: purified hsFANCD2-Ub (2 μM), hsFANCI-Ub (2 μM), hsPCNA-Ub (6 μM), xlFANCD2-Ub (2 μM), and diubiquitin chains (20 μM) were incubated in DUB buffer on ice for 10 min. To initiate hydrolysis, DUBs (200 nM) and substrates (2 or 6 μM) were incubated in a 1:1 ratio (typically 5 μl: 5 μl) for 30 min at room temperature or an indicated time. Reactions were terminated using SDS–PAGE loading buffer (Invitrogen). To analyse deubiquitination, ∼300 ng of FANCD2-Ub or FANCI-Ub, ∼600 ng of PCNA-Ub, or 800 ng of diubiquitin was separated on 4–12% Bis-Tris SDS–PAGE gels and Coomassie stained. Deubiquitination was determined by the loss of ubiquitinated proteins or the emergence of non-ubiquitinated substrates and monoubiquitin. For USP2 DUB assays, the intensity of ubiquitinated substrates was measured using a 700-nm channel after Coomassie staining, calculated as mean % of the input and plotted against time, with error bars showing standard deviation from the mean.

Cells were lysed in 1% Triton X-100 containing protease inhibitors (Complete EDTA-free, Thermo Fisher Scientific, Waltham, MA), 150mM NaCl, and 10% Glycerol for 30 minutes at 4 degrees. Lysates were cleared by centrifugation and protein concentrations determined by BCA Assay (Thermo Fisher Scientific, Waltham, MA). For immunoprecipitation, 1milligram of total protein was incubated with 3 micrograms of antibody overnight at 4 degrees. Protein G agarose beads were added and incubated for 1 hour at 4 degrees then washed 5x in lysis buffer. Beads were boiled in SDS-PAGE loading buffer (Invitrogen) and then resolved on 4–12% Bis-Tris SDS-PAGE gels.

The AAV5 vectors were mixed with SDS-PAGE loading buffer (Invitrogen) and heated at 95°C for 5 min. The vectors were then loaded onto a 10% SDS-PAGE gel and run at 100 volts until the dye reached the bottom of the gel. The gel was stained according to the manufacturer’s protocol (Invitrogen).

To crosslink DUBs with Ub-prg, DUBs (2 μM) in 20 μl were incubated in DUB buffer (50 mM Tris, pH 7.5, 120 mM NaCl, and 10 mM DTT) with and without Ub-prg (6 μM) for 10 min at room temperature. Reactions were stopped by the addition of SDS–PAGE loading buffer (Invitrogen) and visualised using 4–12% Bis-Tris SDS–PAGE (Invitrogen) run with NuPAGE MOPs SDS running buffer (Thermo Fisher Scientific) and Coomassie staining. A slower migrating band indicates reaction with Ub-prg.

Cellular lysates were prepared from exponentially growing cell cultures treated with 40μM camptothecin (Sigma) or 5μg/ml bleomycin (Merck). Denatured cell lysates were prepared by TCA precipitation. Cells were suspended in 20% TCA and lysed mechanically using glass beads (Sigma). Following centrifugation, the TCA precipitate was suspended in SDS-PAGE loading buffer (Invitrogen) containing Tris-base. Protein extracts were directly resolved on Tris-acetate 3–8% polyacrylamide NuPAGE gels (Invitrogen). Proteins were transferred to a nitrocellulose Hybond-C membrane (Invitrogen). The membrane was blocked in PBS-T milk 5% and probed by using anti-Flag (Sigma F1804) antibody (1:5,000 dilution), anti Cdc2 (Santa-Cruz sc-53) antibody (1,1000 dilution), anti-tubulin alpha T5168 (Sigma).