Analysis of the interaction between IRS-1 and HDAC2 in vitro. A: A human foetal brain plasmid cDNA library contained in the p-myr vector was transformed into yeast cdc25 h cells containing full length IRS-1 in the pSos vector. Cell growth at the non-permissive temperature (37°C) on galactose medium (GAL 1 promoter in the library vector) was observed only in the presence of both IRS-1 and HDAC2 from the library from two independent transformants, which both encoded human HDAC2. B: Full length HDAC2 and different sub-cloned domains of IRS- 1 were used in the Matchmaker-3 yeast two-hybrid system to map the interaction between HDAC2 and IRS-1. Growth on medium lacking two nutritional markers was analysed to confirm interactions between predator and prey. C: Recombinant human IRS-1 and HDAC2 were transcribed and translated in vitro individually using a rabbit reticulocyte lysate as described in the Methods section. Proteins were then mixed and IRS-1 was immunoprecipitated from the solution (lane 1). As controls for the immunoprecipitation, the IRS-1 antibody was denatured by boiling prior to immunoprecipitation (lane 2) or all antibodies were omitted and beads alone were used (lane 3). Proteins were resolved by SDS-PAGE, the gel was dried and immunoprecipitated proteins were analysed by phosphorimagery. Protein bands matched IRS-1 (approx 160 kD) and truncated HDAC2 (approx. 35 kD).