Ha beads

Open reading frames of viral proteins were cloned into pcDNA5 mammalian expression plasmid with a C-terminal HA tag. For each replicate, ~150 million 293T cells were transfected with 100 µg of DNA plasmid with calcium phosphate transfection reagents (Clotech). Cells were lysed at 2 days post transfection with binding buffer (50 mM Tris-HCl pH 7.4, 0.5% NP-40, 150 mM KCl, 1 mM EDTA, and protease inhibitors). Cell lysates were incubated with HA beads (Sigma-Aldrich) for overnight at 4 °C with constant agitation, and washed with wash buffer (50 mM Tris-HCl pH 7.4, 2% NP-40, 300 mM KCl, 1 mM EDTA, and protease inhibitors) for five times.

The nuclear fraction was prepared by sequentially lysing the cells with Buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.15% NP40, 1× protease inhibitor (Sigma, P8340)) and Buffer B (20 mM HEPES pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.5% NP40, 1× protease inhibitor). After adjusting the salt concentration to 100 mM with Buffer C (20 mM HEPES, 1× protease inhibitor), the SET-containing protein complex was tandemly precipitated with Flag M2 beads (Sigma, A2220) and HA beads (Sigma, A2095), followed by elution with 0.1% trifluoroacetic acid (TFA, Sigma, T6508-1AMP). After lyophilization, the eluents were redissolved in 1× Laemmli buffer at 95 °C for 5 min, separated by SDS–PAGE and stained with GelCode Blue reagent (Pierce, 24592). The visible bands were isolated and digested with trypsin, followed by liquid chromatography (LC) MS/MS analysis.

Cells for co-IP were lysed in lysis buffer (50 mM Tris, pH 7.5, with 150 mM NaCl, 0.5% NP-40, and protease inhibitor and phosphatase inhibitor cocktail tablets (Roche) at 4 °C for 30 min. After sonication and centrifugation, cell lysates were incubated with HA beads (Sigma) at 4 °C overnight on a rotator. After six washes with wash buffer (20 mM Tris, pH 7.5, 100 mM NaCl, 0.05% Tween-20, 0.1 mM EDTA), 50 μL of elution buffer (100 mM glycine-HCl, pH 2.5) was added to resuspend the beads, and the eluted proteins were obtained by centrifugation, followed by SDS-PAGE and immunoblotting analysis.

Recombinant Emi1 and APC components are purified as previously described (Wang and Kirschner, 2013 (link)). Securin and HA-Nek1 were expressed and radiolabelled with ³⁵S in reticulocyte lysate (Promega). HA-Nek1 was further purified through HA beads (Sigma) for ubiquitylation assay. Cell extracts were prepared from HeLa S3 cells and RPE1 cells as indicated. In vitro protein degradation and in vitro ubiquitylation assays were performed as described previously (Wang and Kirschner, 2013 (link)).

Cells transfected with indicated plasmids were collected and lysed in NETN buffer (10 mM Tris-HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, and 0.5% NP-40) with protease inhibitors (Roche) on ice for 30 min. Then, cell lysates were subject to FLAG M2 (Sigma Aldrich), S-protein beads (Millipore), or HA beads (Sigma Aldrich). After rotation for 8 h at 4 °C, beads were washed with NETN buffer three times, and samples were boiled with 2× sodium dodecyl sulfate (SDS) loading buffer and were subject to immunoblot with indicated antibodies. For endogenous immunoprecipitation (IP), cell lysates were incubated with indicated antibody at 4 °C for 6 h, and then were subject to Protein A/G beads (Thermo fisher) for 4 h at 4 °C. Beads were then washed with NETN buffer three times, and samples were boiled with 2× SDS loading buffer, and were analyzed by immunoblot with indicated antibodies.

HA-FBXW2 or HA- FBXW2-ΔF was respectively purified from HEK293 cells transfected with either plasmid using HA beads (Sigma), and then eluted with 3 × HA peptide (Sigma), serving as the source of E3. FLAG-tagged β-catenin, β-catenin-3A, or β-catenin-3D was respectively pulled down by FLAG beads (Sigma) from HEK293 cells transfected with according plasmid, serving as the substrate. The reaction was carried out at 37 °C for 1 h in 30 μl reaction buffer (40 mM Tris-HCl, pH 7.5, 2 mM ATP, 2 mM DTT, 5 mM MgCl₂) in the presence of E1, E2 and purified E3s. Poly-ubiquitinated β-catenin was resolved by SDS-PAGE, followed by IB with anti-FLAG Ab.