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. 2011 Apr 27;156(2):631–646. doi: 10.1104/pp.111.177667

Figure 5.

Figure 5.

FIN219 can physically interact with COP1. A and B, Interaction of FIN219 and COP1 by the yeast LexA two-hybrid system. Assays of protein-protein interactions are shown by β-galactosidase activities. Values are averages of five individual yeast colonies, and error bars represent sd. The level of activation domain (AD) vector was arbitrarily set to 1. COP1FL, Full-length COP1; COP1-ΔZn, COP1FL minus the zinc-finger domain; COP1-ΔCC, COP1FL minus the coiled-coil domain; COP1-ΔZnΔCC, COP1FL minus the zinc-finger domain and the coiled-coil domain; COP1-N282, the N-terminal 282 amino acid residues of COP1; COP1-WD-40, the WD-40 repeat domain of COP1; COP1FL × HY5FL, a positive control; HY5FL, full-length HY5. Various deletion constructs of FIN219 were described in Figure 4A. DB, DNA-binding domain. C, Interaction of various FIN219 recombinant proteins and full-length COP1 by pull-down assay. Different recombinant proteins, 5 μg each, indicated above the blots were used for pull-down assays. The reaction mixture was precipitated with glutathione-Sepharose 4B beads, and then pellets underwent protein gel-blot analysis with anti-GST or anti-MBP antibodies. Asterisks indicate breakdown products of GST-FIN219FL and GST-FIN219-CT. D, Interaction of various FIN219 recombinant proteins and the WD-40 domain of COP1 by pull-down assay. Asterisks indicate breakdown products of GST-FIN219FL, GST-FIN219-NT, and GST-FIN219-CT. E, Assay of FIN219 and COP1 interaction by BiFC assay. The YN-COP1 and YC-FIN219 plasmids were cotransfected into Arabidopsis mesophyll protoplasts isolated from fin219-null. The transfected protoplasts were cultured overnight in the dark (a–d and i–l) or cFR light (e–h and m–p). F, Coimmunoprecipitation analysis of FIN219 interaction with COP1 in the wild type (Col). Two milligrams of protein extracts isolated from Col and fin219-2 seedlings grown in cFR light for 3 d was mixed with the FIN219 polyclonal antibodies and then coimmunoprecipitated. The pellets were used for SDS-PAGE and protein gel-blot analysis. The probes are FIN219 and COP1 antibodies. I, Input proteins; S, supernatants after precipitation; P, pellets after precipitation. The double asterisk indicates artificial bands.