Tnt in vitro translation kit

The yeast strain EGY48 containing pSH18-34 (8XlexA_op-LacZ reporter plasmid) was co-transformed with expression plasmids for LexA-fused bait and B42-GBD-fused prey by the lithium acetate method. Liquid assays for β-galactosidase activity were carried out for three transformants as described previously (25 (link)). HA-tagged wild-type HDAC4c/-5c was labeled with ³⁵S-methionine using a TNT in vitro translation kit (Promega, Madison, WI, USA). The radiolabeled proteins were mixed with equivalent amounts of GST or GST-fused proteins bound to glutathione-agarose beads (Sigma-Aldrich, St. Louis, MO, USA) pre-equilibrated with GST binding buffer [25 mM HEPES–KOH (pH 7.5), 10% glycerol, 200 mM KCl, 1 mM EDTA, 0.01% Nonidet P-40, 1 mM DTT, 1× protease inhibitor cocktail] in a final volume of 300 μl. The beads were washed three times with the same buffer, and bound proteins were analyzed by 8% SDS-PAGE followed by autoradiography. Details of the expression and purification of GST proteins have been described previously (20 (link)).