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. 2014 Jun 11;165(4):1737–1750. doi: 10.1104/pp.114.236448

Figure 4.

Figure 4.

NtCDPK1 largely colocalizes with RSG and 14-3-3. Constructs expressing fluorescent protein fusion proteins were cotransfected into Arabidopsis T87 protoplasts. AGF1-CrFP was used as a control for nuclear localization. After 24 h, the cells were visualized by confocal laser scanning microscopy. Fluorescence intensities were quantified by ZEN 2009 software (Carl Zeiss). Distance in the graph of fluorescence intensity corresponds to the position of the arrow (from nock to head) in the merged image. This experiment was repeated three times with similar results. Bars = 5 µm. A, 14-3-3 colocalizes with the NtCDPK1-RSG complex in the cytoplasm. Shown are the reconstituted YFP fluorescence resulting from the interaction between YFPN-NtCDPK1 and RSG-YFPC (top left), the mRFP fluorescence of mRFP-14-3-3 (top second from left), the CrFP fluorescence of AGF1-CrFP (top second from right), the bright-field image (top right), the merged image (bottom left), and the quantified fluorescence intensities (bottom right). Pinhole settings are 2.78 airy units (AU) for YFP, 2.43 AU for mRFP, and 2.86 AU for CrFP. B, NtCDPK1 colocalizes with the 14-3-3-RSG complex in the cytoplasm. Shown are the reconstituted YFP fluorescence resulting from the interaction between YFPN-14-3-3 and RSG-YFPC (top left), the mRFP fluorescence of mRFP-NtCDPK1 (top second from left), the CrFP fluorescence of AGF1-CrFP (top second from right), the bright-field image (top right), the merged image (bottom left), and the quantified fluorescence intensities (bottom right). Pinhole settings are 1 AU for YFP, 1 AU for mRFP, and 1 AU for CrFP. C, RSG colocalizes with the 14-3-3-NtCDPK1 complex in the cytoplasm. Shown are the reconstituted YFP fluorescence resulting from the interaction between YFPN-14-3-3 and YFPC-NtCDPK1 (top left), the mRFP fluorescence of RSG-mRFP (top second from left), the CrFP fluorescence of AGF1-CrFP (top second from right), the bright-field image (top right), the merged image (bottom left), and the quantified fluorescence intensities (bottom right). Pinhole settings are 1.48 AU for YFP, 1.43 AU for mRFP, and 1.25 AU for CrFP. [See online article for color version of this figure.]