Fluorimetric Analysis of CIPK Proteins

Fluorimetric analyses were performed in yeast as previously described [15 (link)]. In brief, CIPK15, CIPK19, and CIPK15m (K41N, an inactive form) were introduced into yeast expressing AmTryoshka1;3 LS-F138I. Vector only was used as the control. Cells were analyzed in 96-well, flat-bottom plates (Greiner Bio-One, Germany). Steady-state fluorescence was recorded using a fluorescence microplate reader (Infinite, M1000 pro, Tecan, Switzerland) in bottom-reading mode using 7.5 nm bandwidth and a gain of 100. The fluorescence emission spectra (λ_exc 440 or 485 nm; λ_em 510 or 570 nm) were background-subtracted using yeast cells expressing a non-florescent vector control.