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Figure S1. CBL4::GFP and CBL8 ::GFP, but not CBL7::GFP, partially co-localize with the PM marker CBL1n::OFP. in transiently transformed Nicotiana benthamiana leaves. OFP channel, left, GFP channel, middle; overlay, right. A plot shows the distribution of all pixels throughout the figure. In CBL4::GFP + CBL1n::OFP the pixels are distributed on the same linear slope indicating co-localization of the pixels. Partially, this is also observed for CBL8::GFP + CBL1n::OFP. In contrast, in CBL7::GFP + CBL1n::OFP the green and red pixels are clearly separated.

Figure S2. CBL4::GFP and CBL5::GFP do not co-localize with the tonoplast bound protein CBL3::OFP in transiently transformed Nicotiana benthamiana leaves. GFP channel, left; OFP channel, middle; overlay, right. Additionally, the distribution of green and red fluorescence were analyzed by scanning fluorescence intensity at the given ‘region of interest’ (arrow).

Figure S3. (a) Co-localization of CBL2::GFP, CBL3::GFP, CBL6::GFP with TPC1::OFP. The distribution of green and red fluorescence were analyzed by scanning fluorescence intensity at the given ‘region of interest’ (arrow). (b) Additionally, co-localization studies were performed with CBL3::GFP and OFP (wt protein) and the PM marker CBL1n::OFP.

Figure S4. CBL2::GFP co-localizes with OFP fusions of CBL2, CBL3, CBL6 and CBL10 at the vacuolar membrane of transiently transformed Nicotiana benthamiana leaves. GFP channel, left; OFP channel, middle; overlay, right.

Figure S5. Alignment of the Arabidopsis CBL N-termini. Gap formation was allowed for CBL6 and CBL7. Identical amino acids are decorated by a black box. Bold letters indicate amino acids which were fused with GFP (CBLxn::GFP contructs).

Figure S6. (a) CBL7::GFP and CBL8::GFP infiltrated with different densities of Agrobacteria. The upper row shows a Western Blot using a specific GFP antibody, of a total protein extract, prepared from Nicotiana benthamiana leaves infiltrated with the respective density of Agrobacteria (O.D.600 = 0.05, 0.1 and 0.5) (7 = CBL7::GFP; 8 = CBL8::GFP; c = control). The lower row depicts confocal figures taken from the same leaf. Laser and scanning settings were adjusted to the fluorescence from the leaf infiltrated with the highest density of Agrobacteria. (b) Expression and localization of CBL8::GFP at different timepoints. The upper row shows a Western Blot of a total protein extract using a specific GFP antibody, extracted from samples at the indicated time points (h = hours; d = days). For confocal imaging, laser and scanning settings were adjusted to the fluorescence in the leaf incubated either for three (upper row) or 5 days (lower row). c = control.

Figure S7. CBL7::GFP co-localizes with OFP but not with OFP::HDEL (ER marker), GNT1::OFP (Golgi marker) or CBL3::OFP (tonoplast marker).

Figure S8. Schematic representation of CBL3, CBL4, CBL5, CBL7, CBL8, CBL9 and CBL10 proteins and of CBLn and CBL&Dgr;n GFP fusions. Displayed are the CBL EF-hands (numbered boxes) and the N-terminal fragments (grey) which were used for GFP fusion (green). N-terminal lipid modifications (jagged line), the transmembrane domain of CBL10 (TMD) and the size of the CBL proteins, of the CBL N-termini and the position of the N-terminal deletion in CBL10 are indicated.

Figure S9. CBL10 contains an N-terminal transmembrane domain. (a) For CBL10, a transmembrane domain is predicted by the TMHMM server (v.2.0; blue line) and by the SOSUI server (v.1.11; grey box; aa 17–35) which fits to the hydropathy plot from the DAS-TMfilter. No transmembrane domain is predicted for CBL2 (green and turquoise line). The relative positions of the EF-hand loops are depicted in the graph. (b) The majority of servers predict the start of the CBL10 transmembrane domain at aa 17 and the end at aa 35/36. The length of the transmembrane domain is estimated to be 19–20 aa.

Figure S10. Co-localization of GFP fused with the N-terminus of CBL10 (GFP::CBL10n) or CBL10 lacking the putative TMD (CBL10&Dgr;TMD::GFP) with either OFP (wt protein) or the PM marker CBL1n::OFP. GFP channel, left; OFP channel, middle; overlay, right.

Figure S11. Co-localization analysis of CBL2::SPYCE + CIPK14::SPYNE and CBL3::SPYCE + CIPK14::SPYNE with TPC1::OFP, and CBL3::SPYCE + CIPK14::SPYNE or CBL8::SPYNE + SPYNE::CIPK14 with CBL1n::OFP. The distribution of yellow fluorescence (here presented as green colour) and red fluorescence were analyzed by scanning fluorescence intensity at the given ‘region of interest’ (arrow).

Table S1. Constructs published previously and primers used in this work.

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TPJ_4045_sm_figures.pdf11346KSupporting info item
TPJ_4045_sm_tables.pdf55KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.