Bortezomib

HA-tagged PGC-1α was co-transfected into HEK-293FT cells with GCN5 for 48 h and the cells were lysed in RIPA lysis buffer supplemented with complete protease inhibitor cocktail (Roche), 1 mM phenylmethylsulphonylflouride (PMSF), 4 μM TSA (Cell Signaling), 5 mM nicotinamide, and 1 μM Bortezomib (Cell Signaling) on ice. Lysate was sonicated, cleared by centrifugation, and immunoprecipitation of acetylated PGC-1α performed with anti-HA agarose beads (Pierce) performed at 4°C with rotation in the presence of inhibitors. Sequential washes (with inhibitors) were performed: six washes with RIPA buffer, two washes high salt wash buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% Sodium-deoxycholate, 1 mM EDTA), followed by two washes in 1x PBS. The purified protein remained immobilized on HA-beads in the presence of inhibitors prior to de-acetylation assay.

For cellular acetylation experiments, 293FT cells were co-transfected with combinations of pcDNA3.1-GFP, pcDNA3.1-HA-PGC-1α, pcDNA3.1-HDAC3-myc, pcDNA3.1-Flag-GCN5, and/or empty pcDNA3.1. 48 h after transfection, cells were harvested in ice-cold NP40 IP Lysis Buffer (20 mM Tris-HCl, 150 mM NaCl, 10% glycerol, 2 mM EDTA, 0.1% NP40, 10 mM NaF, with 1 mM DTT, 1 mM phenylmethylsulphonylflouride (PMSF), and supplemented with complete protease inhibitor cocktail (Roche), 4 μM Trichostatin A (TSA, Cell Signaling), 5 mM nicotinamide (Sigma), and 1 μM Bortezomib (Cell Signaling) on ice. Cell lysates were freeze/thawed in liquid nitrogen twice, and lysates cleared by centrifugation at 4°C for 10 minutes. 250 mg of protein was used for each immunoprecipitation and volume adjusted to 1ml with lysis buffer. Washed anti-HA Agarose beads (Pierce) were incubated with samples and rotation at 4°C for 4 h, followed by three brief washes with lysis buffer. Immunoprecipitated proteins were eluted in 2X loading buffer with boiling at 95°C for 5 minutes.