Figure 3.

Effects of overexpression of pho8 and pho2 on alkaline phosphatase activity. A, immunoblot analysis of protein extracts prepared from pho8Δ cells expressing the indicated fusion proteins following growth in ZL-EMM supplemented with 0, 1, 10, or 100 μm zinc or the empty vector following growth in ZL-EMM and 100 μm zinc. Protein extracts prepared from pho8Δ cells expressing pgk1-Pho8-GFP were diluted 5-fold. The positions of the molecular mass markers in kilodaltons are shown on the left, and the arrow indicates the full-length Pho8-GFP protein. Bottom panel, the mean ratio of Pho8-GFP/Act1 levels from three independent repeats, with error bars representing standard deviations. **, p < 0.01; ns, not significant. B, immunoblot analysis was performed as described in A with the indicated strains. C, WT, pho2Δ, and pho2Δ pho8Δ cells bearing the empty vector and pho8Δ and pho2Δ pho8Δ cells expressing Pho8-GFP were assayed for alkaline phosphatase (ALP) activity following growth overnight in ZL-EMM supplemented with 0, 1, 10, or 100 μm zinc. Activity is the mean of three independent repeats, with the error bars representing standard deviations. D, WT, pho2Δ, and pho2Δ pho8Δ cells bearing the empty vector, and pho2Δ, and pho2Δ pho8Δ cells expressing Pho2-GFP were grown and assayed for alkaline phosphatase activity as described in C.