Fig 2. Expression of the ZapCY1 and ZapCY2 FRET reporters in fission yeast.

(A) Schematic diagram of the ZapCY1 FRET sensor. Zinc fingers are shown as gray cylinders, and YFP and CFP are shown as yellow and blue colored stars. (B) Wild-type cells expressing ZapCY1, ZapCY2, or the empty vector were grown overnight in ZL-EMM without (-Zn) or with a 100 μM Zn²⁺ supplement (+Zn). Cells were collected and crude protein extracts prepped for immunoblot analysis. Immunoblots were probed with antibodies raised against GFP and the loading control Act1 (Actin). A protein ladder with sizes in kDa is shown on the left (C) Wild-type cells expressing the indicated reporters or the vector were grown in ZL-EMM (-Zn) or ZL-EMM + 100 μM Zn²⁺ (+Zn) and were analyzed by fluorescence microscopy (GFP). Bright field images are also shown. (D) Wild-type cells expressing ZapCY1, ZapCY2, or an empty vector (vec) were grown in ZL-EMM supplemented with 0, 1, 10, or 100 μM Zn²⁺ and total RNA purified for RNA blot analysis. RNA blots were probed for the zinc-regulated zrt1 and zym1 transcripts, and loading control pgk1. (E) β-galactosidase activity was measured in wild-type cells co-expressing a zrt1-lacZ reporter with the empty vector, ZapCY1, or ZapCY2. Cells were grown as described in panel D. Each bar shows the average value from three independent experiments and error bars show the standard deviations.