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. 2014 Sep 8;166(3):1312–1328. doi: 10.1104/pp.114.242487

Figure 2.

Figure 2.

CP is present on cytoplasmic puncta that display only modest colocalization with actin filaments or cables in epidermal pavement cells. Seedlings of wild-type Arabidopsis plants (20 DAG) were fixed and prepared by the freeze-shattering method prior to incubation with affinity-purified CPA or CPB polyclonal antisera, as well as with a mouse monoclonal IgM against actin. Epidermal pavement cells were examined by confocal laser scanning microscopy and images shown are z-series projections. A, The left image shows a control with secondary antibody only (i.e. no CP primary antibody). The middle image shows actin labeling and the right image is a color overlay of the control (green) and actin (red) images. B, A representative epidermal pavement cell that is double labeled for CPA (left) and actin (middle). The right image is a color overlay of CPA (green) and actin (red). CPA is present on cytoplasmic puncta or foci of varying size and intensity. A small subset of these colocalize (right, yellow) with actin filaments or cables. C, A representative epidermal cell that is double labeled for CPB (left) and actin (middle). The right image is a color overlay of CPB (green) and actin (red). Similar to CPA, CPB is present on puncta that sometimes colocalize (yellow) with actin cables. D, Colocalization of Golgi and actin filaments. Arabidopsis seedlings expressing the Golgi marker mannosidase-YFP were prepared and immunolabeled as above with the actin monoclonal antibody. The left image shows mannosidase-YFP fluorescence and the middle image is actin. The right image is a color overlay of mannosidase-YFP (green) and actin (red), showing a substantial overlap (yellow) of Golgi on the actin cables (yellow). E, Quantitative analysis of CPA, CPB, and mannosidase-YFP association with actin filaments and cables. See “Materials and Methods” for details. The mean values (± sem) from analysis of more than 25 ROIs per treatment are plotted. Compared with controls, in which the CP primary antibody was excluded, the extent of colocalization between CPA, CPB, or mannosidase-YFP with actin was significant (*P < 0.01). CTRL, Control; Mann, mannosidase.