Analysis of conditions for interaction between IRS-1 and HDAC2 in mammalian cells and tissues. A: MCF-7 human breast carcinoma cells were treated with 10 ng/ml IGF-1 ("I") or 10 ng/ml PMA for the indicated times. IRS-1 was immunoprecipitated from cell lysates and western blots were analysed for co-immunoprecipitation of HDAC2. The upper gel shows presence of HDAC2 whilst the lower gel is a loading control for IRS-1. The data are representative of multiple experiments. Similar data are obtained if IGF-1 is exchanged for 100 nM insulin. B: MCF-7 cells were treated PMA (10 ng/ml) or TSA (150 ng/ml) for 4 hours prior to stimulation with or without insulin (100 nM) for 10 minutes, lysis and immunoprecipitation of IRS-1. Western blots were probed for the presence of phospho-serine 312 in IRS-1 (upper gel) and IRS-1 (lower gel). The histogram shows the means ± range for results for serine 312 phosphorylation from two independent experiments, normalised to the maximum phosphorylation signal, which was seen in cells treated with PMA and TSA. In separate control experiments, we have seen that the phosphorylation of serine 312 in cells is driven by PMA and that TSA does not contribute to the effect (data not shown). C: Liver tissues from ob/ob mice, c57/bl6 mice, PTP1B knockout mice and balb/cjj mice were prepared as described in the Methods section and IRS-1 was immunoprecipitated. Western blots were analysed for the presence of HDAC2 (upper gel) or IRS-1 (lower gel). Each lane is from one mouse and is representative of at least two other animals per group.