Human recombinant gst e1

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 GST-E1 (50 nM, Boston, Biochem, Cambridge, MA, Cat.#E-306), human recombinant UbcH5c/UBE2D3 (2.5 μM, Boston Biochem, Inc., Cambridge, MA, Cat.#E2-627), human recombinant ubiquitin (250 μM, Boston Biochem, Inc., Cat.#U-100H), human MuRF3 recombinant protein (1 mg, LifeSensors, Cat.#UB306, Malvern, PA), human PPAR-α, −β, and -γ recombinant protein (500 ng, Sigma-Aldrich, Inc., St. Louis, MO, 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-MuRF3 antibody (Cat.#sc-50252, Santa Cruz Biotechnology), rabbit polyclonal anti-PPARα antibody (Cat.#Ab24509, Abcam), rabbit polyclonal anti-PPARβ antibody (Cat.#AB10094, Millipore), or rabbit polyclonal anti-PPARγ antibody (Cat.#2443, Cell Signaling Technology).