4.4. Protein Isolation and Western Blotting

For whole cell protein isolation, liver tissue or primary hepatocytes were homogenised and lysed in 1× RIPA buffer (50 mM TrisHCl pH 8, 150 mM, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 1 mM NaVO₃, 1 mM DTT, 1 mM PMSF) containing protease inhibitor cocktail solution. Protein lysates were stored at −80 °C until analysed. For nuclear and cytoplasmic protein isolation, liver tissue was homogenised using Dignam A lysis buffer (10 mM HEPES pH 7.9, 1.5 mM MgCl₂, 10 mM KCL, 50 mM PMSF, 1 M DTT and protease inhibitor) and centrifuged. The supernatant and the pellet were collected separately. The supernatant consists the cytoplasmic protein fraction, which was stored at −80 °C. For nuclear protein fraction, the pellet was washed in Dignam B buffer (Dignam A with 0.1% Triton X-100) and afterwards re-suspended in Dignam C buffer (20 mM HEPES pH 7.9, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl₂ and 0.2 mM EDTA, 1 mM DTT, 1 mM PMSF, 1 mM Na₃VO₄ and protease inhibitor) and stored at −80 °C until analysed. Bradford assay was used to measure protein concentration. Standard western blotting protocol was adopted for western blot experiment.