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. Author manuscript; available in PMC: 2014 Jun 27.
Published in final edited form as: Methods Mol Biol. 2014;1062:629–658. doi: 10.1007/978-1-62703-580-4_33

Figure 2.

Figure 2

BiFC analyses in Nicotiana benthamiana and Arabidopsis. (a) Representative images of YN-CIPK24/CBL10-YC BiFC (yellow, tonoplast) co-expressed with SCFP3A (cyan, cytoplasm and nucleus) in N. benthamiana. Left panel, overview images and right panel, maximum projections of a z-stack including 32 focal planes of the same cell (60× objective, scale = 20 μm). (b) Normalized YFP fluorescence (BiFC) or BiFC/SCFP3A (Y/C)-ratios from randomly taken images of epidermal cells infiltrated with the indicated BiFC construct combinations using a 20× objective 3 days after infiltration into N. benthamiana. Given are averages ± standard errors (n = 10). Compared to CIPK24/CBL10, expression of CIPK24NAFΔ/CBL10 exhibited only 41 – 57 % BiFC. (c) Western blot analyses of BiFC construct/SCFP3A combinations from the same experiments as in (a and b). From top to bottom, CBL10-YC detected with α-HA antibody, YN-CIPK24(NAFΔ) detected with α-GFP antibody, SCFP3A detected with α-GFP antibody and PageBlue staining of the large RuBisCO subunit (rbcL) in representative SDS-gel as loading control. Loaded were 10 μg total protein for CBL10 and rbcL and 15 μg for CIPK24(NAFΔ) and SCFP3A blots. In the p19 lane protein extracts of only with p19 infiltrated leaves were loaded. Western blots confirm expression of BiFC and SCFP3A constructs in indicated construct combinations. (d) BiFC analyses of YN-OST1/YC-ABI1 in N. benthamiana (N.b.; left panel) and Arabidopsis (A.t.; right panel). Depicted are overview images (left) and maximum projections of 32 focal plane z-stacks (right). Images were taken with a 60× objective 4 (N.b.) and 7 days (A.t.) after infiltration. Scalebars = 20 μm. BiFC analyses indicate OST1/ABI1 complex formations in the cytoplasm and the nucleus.