N ethylmaleimide

Cells were lysed with radioimmunoprecipitation assay buffer containing 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 0.1% sodium dodecyl sulfate (SDS), 0.25% sodium deoxycholate, 10 mM iodoacetamide (Sigma-Aldrich), 10 mM N-ethylmaleimide (Sigma-Aldrich), 0.5 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF), 10 μM MG132, and PhosStop phosphatase inhibitors (Sigma-Aldrich). Endogenous Cyclin A2, TFIIB, PRPS1/2/3, CUL5, ACSL4, PCNA, DHPS, and BLVRA were immunoprecipitated with an anti-Cyclin A2 antibody (B-8, sc-271682, SCBT), anti-TFIIB antibody (D-3, sc-271736, SCBT), anti-PRPS1/2/3 antibody (A-11, sc-376440, SCBT), anti-CUL5 antibody (sc-13014, SCBT), anti-ACSL4 antibody (22401-1-AP, Proteintech), anti-PCNA antibody (PC10, sc-56, SCBT), anti-DHPS antibody (A-10, sc-365077, SCBT), and anti-BLVRA antibody (F-1, sc-393385, SCBT) respectively and immunoblotted. Alternatively, His₆-ubiquitin was introduced into cells and the cells were lysed with a buffer containing 50 mM Tris-HCl (pH 7.6), 300 mM NaCl, 8 M urea, 0.5% Triton X-100, 10 mM iodoacetamide (Sigma-Aldrich), 10 mM N-ethylmaleimide (Sigma-Aldrich), 0.5 mM AEBSF, 10 μM MG132, and PhosStop phosphatase inhibitors (Sigma-Aldrich). His₆-ubiquitin-conjugated proteins were pulled down with Probond resin (Thermo Scientific) and immunoblotted with several antibodies.

Yeast strains from the deletion collection were a gift from Claire Moore (Tufts University), rsp5-1 was a gift from Fred Winston (Harvard Medical School). Yeast strains were grown in YPD (Sigma). Yeast were grown to log phase, frozen in liquid nitrogen, and thawed as needed. The extract was prepared fresh by bead beating in 20 mM Tris pH 7.9, 50 mM KCl, 0.5 mM DTT, 10% glycerol, with PMSF and N-ethylmaleimide (Sigma).
Hela and human lens epithelial cells were maintained in DMEM supplemented with 10% FBS and penicillin/streptomycin. Transfections were carried out in OptiMEM medium using Lipofectamine 2000 or Lipofectamine 3000 according to the manufacturer’s directions. Smurf1 and Smurf2 RNAi were from Dharmacon, UbcH7 RNAi from Qiagen. Extracts were prepared in RIPA buffer (Thermo Scientific) supplemented with protease inhibitor cocktail (Sigma) and N-ethylmaleimide (Sigma). Nedd4 family ligase expression vectors were a kind gift from Dr Wesley Sundquist (University of Utah). GST-Rsp5 and GST-Rsp5 C777A were a gift from Dr Joseph Reese (Penn State).

Cells were collected, washed with PBS, lysed with RIPA lysis buffer containing 10 mM N-ethylmaleimide (EMD Millipore) and 1 mM PMSF, and sonicated on ice for protein extraction. After sonication, the cell lysates were centrifuged at 13,000 × g and 4 °C for 15 min. The supernatants were incubated with 1 μg anti-IκB antibody at 4 °C overnight, and they were then attached to 20 μL of Protein G agarose beads by mixing on a rotator at 4 °C for 2 h. Cell lysates were washed with RIPA lysis buffer containing 10 mM N-ethylmaleimide and 1 mM PMSF (Sigma-Aldrich, St. Louis, MO, USA), and they were boiled in 2× sample buffer for 10 min. The protein–protein interactions were verified via western blot analysis.

The biotin switch assay was performed as previously described (Hemsley, Taylor, & Grierson, 2008 (link)) with minor modification. Briefly, about 0.8 mg total proteins were solubilized and incubated with 25 mM N‐ethylmaleimide (Thermo Fisher Scientific) to block free sulfhydryls. Free N‐ethylmaleimide was removed by concentration tube (Millipore) then divide into two equal aliquots. One aliquot was incubated with 1 M hydroxylamine (NH₂OH) (Thermo Fisher Scientific) to cleave thioester bonds and with 1 mM EZ‐link™ biotin‐HPDP (Thermo Fisher Scientific) to label liberated sulfhydryls. Hydroxylamine was replaced by Tris‐HCl buffer in the remaining aliquot as a control. Free reagent was removed as mentioned above, and 12 μl of the solution was removed as a loading control. Biotinylated proteins were then purified with 15 μl NeutrAvidin‐agarose (Thermo Fisher Scientific) and analyzed by Western blotting using GFP‐specific antibodies.

Cell lysates were incubated with the indicated antibodies at 4 °C overnight and added with 50 μl Protein A/G Sepharose beads (Santa Cruz) for another 4 h. The immunoprecipitates were washed and then processed for western blotting.
For the sequential IP assay, lysates from HEK-293 cells transfected with HA-GSK3β, Myc-Twa1 and Flag-Axin were incubated with anti-Flag antibody bound to protein A/G-agarose beads at 4 °C overnight. The immunoprecipitates were washed and then eluted with Flag peptide for 2 h at 4 °C. The Flag eluates were subsequently incubated with anti-Myc antibody or control IgG at 4 °C overnight and added with protein A/G-agarose beads for another 4 h. The immunoprecipitates were washed and then processed for western blotting.
For the ubiquitination assays, the cells were treated with 25 μM MG132 (Sigma) for 2 h before collection and lysed by RIPA buffer with 10 mM N-ethylmaleimide (Sigma) and a cocktail of protease inhibitors. The prepared lysates were then immunoprecipitated with anti-Flag antibody at 4 °C overnight and subjected to western analysis.