Recombinant human ubiquitin

Endogenous mRNAs in thawed extracts were degraded by treatment with 0.3 U/μl micrococcal nuclease (MNase) and 480 μM CaCl₂ for 10 min at room temperature, followed by addition of 2 mM EGTA and transfer to ice. ScIVT reactions were initiated by adding m⁷G-capped RNA (40 ng per μl of reaction volume) to MNase-treated yeast extracts and incubating at 25°C for up to 90 min. Final concentrations of reaction components were 48.67% (v/v) MNase-treated yeast extract, 22 mM HEPES-KOH (pH 7.4), 120 mM potassium acetate, 1.5 mM magnesium acetate, 0.75 mM ATP, 0.1 mM GTP, 0.04 mM each amino acid, 1.7 mM DTT, 25 mM creatine phosphate, 0.34 μg/μl creatine kinase, 0.14 U/μl SUPERase•In RNase Inhibitor (Thermo Fisher Scientific), and 0.16X cOmplete mini EDTA-free protease inhibitor cocktail (Roche). Where indicated, reactions also included 10 or 100 μM recombinant human ubiquitin or Myc-ubiquitin (Boston Biochem, Cambridge, MA). Reactions were halted by transferring to ice or by adding an equal volume of 2X Laemmli Sample Buffer (Bio-Rad). The results shown for all ScIVT experiments are representative of at least two technical replicates (i.e., experiments conducted with independently prepared reagents).

Human TOP1 was purified from baculovirus as described^{69 (link)}. Recombinant human ubiquitin was purchased from R&D Systems (Cat. # U-100H). Recombinant human ubiquitin-activating enzyme E1 (UBE1) was purchased from R&D systems (Cat. # E-305). Recombinant human UbcH5a was purchased from R&D systems (Cat. # E2-616). For purification of the CUL4A-RBX1 complex, synonymously mutated His-Cul4a was co-expressed with His-Rbx1Δ1–14 in SF9 cells. Cells were resuspended in buffer containing 50 mM Tris, 200 mM NaCl, 0.1% Triton X-100, 1 mM TCEP, and 1x SigmaFAST Protease Inhibitors, pH 8.0. Cell suspension was sonicated for 12 cycles at 10 s per cycle, with 30 s of cooling between cycles at 4 °C. Lysate was centrifuged at 20,000 × g for 30 min, and incubated with Ni-NTA agarose for 1 h with rotation at 4 °C. Beads were washed with wash buffer (50 mM Tris, 200 mM NaCl, 10 mM imidazole, 1 mM TCEP, pH 8.0) and eluted with elution buffer (50 mM Tris, 200 mM NaCl, 300 mM imidazole, 1 mM TCEP, pH 8.0). Eluate was injected onto a Superdex200 Increase 10/300 column (GE Healthcare), and the protein complex eluted at 13.8 ml. Protein was concentrated and stored at −80 °C in 50 mM Tris, 200 mM NaCl, 1 mM TCEP, pH 8.0.

Human recombinant GST-E1 (50 nM, Boston, Biochem, Cambridge, MA, Cat. #E-306), human recombinant UbcH5c/UBE2D3 (2.5 μM, Boston Biochem, Inc., Cambridge, MA, USA, Cat. #E2-627), human recombinant ubiquitin (250 μM, Boston Biochem, Inc., Cat. #U-100H), human MuRF2 recombinant protein (1 mg, LifeSensors, Cat. #UB305, Malvern, PA, USA), human PPAR-α, -β, and -γ recombinant protein (500 ng, Sigma-Aldrich, Inc., St. Louis, MO, USA, Cat. #SRP2043, Cat. #SRP2044, and Cat. #SRP2045, respectively) were added to reaction buffer (50 mM HEPES, pH 7.5) containing 5 mM MgATP solution (Boston Biochem, Inc., Cat. #B-20) and 0.6 mM DTT then incubated at 37°C for 1 h. The reaction was stopped by adding SDS-PAGE sample buffer and heating, then resolved on a 4–12% Bis–Tris gel with MOPS running buffer (Invitrogen Corp.) and transferred to PVDF membranes for immunoblotting with goat polyclonal anti-MuRF2 antibody (Abcam, Cat. #Ab4387), rabbit polyclonal anti-PPARα antibody (Abcam, Cat. #Ab24509), rabbit polyclonal anti-PPARβ antibody (Millipore, Cat. #AB10094), or rabbit polyclonal anti-PPARγ antibody (Cell Signaling Technology, Cat. #2443).

Human recombinant Top1 baculovirus-infected insect cells in media were centrifuged at 2,000 × g and the pellet was resuspended in ice-cold lysis buffer (25 mM Tris pH 7.5, 100 mM NaCl, 5 mM EDTA, 0.5% NP40, 4 mM β-mercaptoethanol, and 10 mM PMSF) and then incubated on ice for 10 min. The sample was then centrifuged at 6,000 × g for 20 min and the supernatant was then dialyzed against 25 mM Tris pH 7.5, and 4 mM β-mercaptoethanol to remove the EDTA. TOP1 was purified on a HiTrap Q HP column (Amersham Biosciences, Uppsala, Sweden) in buffer A (25 mM Tris pH 7.5, 5 mM β-mercaptoethanol, 5 mM EDTA) and eluted with buffer B (buffer A with 1 M KCl). The TOP1 peak fraction was dialyzed against buffer A, made 50% glycerol, and stored at −20 °C^{49 (link)}. Human recombinant PARP1 was purified from E. coli as described^{50 (link)}. Human recombinant USP7 was purchased from R&D Systems (cat # E-519). Human recombinant ubiquitin was purchased from Boston Biochem (cat # U-100H). Human recombinant ubiquitin Activating Enzyme (UBE1) was purchased from Boston Biochem (cat # E-305). Human recombinant UbcH5a/UBE2D1 was purchased from Boston Biochem (cat # E2-616). Human recombinant RNF4 was purchased from R&D Systems (cat # E3-210-050).