FIGURE 4.

Blocking lanosterol production reduced Hmg2-Nsg1 interaction. A, the Hmg2-Nsg1 interaction was disrupted when sterol synthesis was blocked. WT and nsg1Δ cells were treated with Tb (10 μg/ml) and MG132 (25 μg/ml) or with MG132 alone for 2 h at 30 °C. 10 OD equivalents of cells for each sample were lysed in non-denaturing TMM, microsomes were prepared and then solubilized with 1% digitonin, and then Nsg1–3HA was immunoprecipitated (IP) by anti-HA affinity matrix. 1mycHmg2 and Nsg1–3HA were detected by immunoblotting. Similar experiments were repeated at least five times with similar results. B, specifically blocking lanosterol production disrupted the Hmg2-Nsg1 interaction. WT and nsg1Δ cells were treated with the series of pathway inhibitors and MG132 (25 μg/ml) or untreated (MG132 only) for 2 h at 30 °C. The co-immunoprecipitation assay was performed as in A. Inhibitors were used at the same concentration as in Fig. 2A. The experiment was repeated at least three times with similar results.