Anti adme r

Total protein from cells and liver tissue was extracted in ice-cold radioimmunoprecipitation assay buffer (RIPA buffer) (150 mM NaCl, 50 mM Tris-HCl pH 8.0, 5 mM EDTA, 0.5% sodium deoxycholate, 0.1% SDS, 1% NP-40) supplemented with protease inhibitor cocktail (Sigma-Aldrich). Protein concentration was measured by DC protein assay reagents (Bio-Rad Laboratories) in SpectraMax M3 multi-mode microplate reader (Molecular Devices). Protein lysate was subjected to SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. After incubation with blocking buffer (5% nonfat milk in 1% TBS with Tween 20) for 1 hour, the membranes were probed with primary anti-PRMT1 (Cell Signaling Technology, catalog 2449S), anti-HSP90 (Cell Signaling Technology, catalog 4874S), anti-HA (Cell Signaling Technology, catalog 3724), anti-α-tubulin (Cell Signaling Technology, catalog 2144), anti-Adme-R (Cell Signaling Technology, catalog 13522), anti-pAkt (Cell Signaling Technology, catalog 9271S), anti-Akt (Cell Signaling Technology, catalog 9272), and anti-Histone H3 (Active motif, catalog 39763). Secondary antibody linked with horseradish peroxidase was diluted in 5% nonfat milk in 1% TBS with Tween 20 and incubated for 2 hours at room temperature. The blots were developed by ECL (Bio-Rad Laboratories, 1705061). Quantification of immunoblot analyses was performed using Quantity One (Bio-Rad).