AtMPK6 interacted with AGB1 in Y2H, Pull-down, BiFC and Co-IP assays.

(A) Interaction tests using yeast two-hybrid assays between AGB1 and AtMPK6. Yeast cells with AGB1 (AD-AGB1) and AtMPK6 (BD-MPK6) were placed in different liquid concentrations on control medium SD/-Trp/-Leu (SD/LT) and selection medium SD/-Trp/-Leu/-His/-Ade (SD/LTHA). For negative controls, pGBKT7 without insert (BD alone), pGADT7 without insert (AD alone), and the empty vectors AD and BD were used. Experiments were performed three times and a representative result is shown. (B) AGB1 interacted with AtMPK6 by BiFC assays in Arabidopsis protoplast. The recombinant constructs AGB1-YFP^N (YFP N-terminal) and MPK6-YFP^C (YFP C-terminal) were co-transformed into protoplast cells of Arabidopsis WT (Col-0). For negative controls, pSPYNE without insert AGB1 (YFP^N) (iii) and pSPYCE without insert MPK6 (YFP^C) (ii) were used. The combination of bZIP63-YFP^N and bZIP63-YFP^C was used as a positive control (iiii), Fluorescence was recorded 20 h after transformation. Experiments were performed 10 times and a representative result is shown. Bars = 40 μm in (i), (iii) and (iiii), and 20 μm in (ii). (C) Interaction assay using pull-down analysis. AGB1 and AtMPK6 fused with GST and His tags, respectively were mixed and passed through a glutathione column (binding GST tag). After elution with pull-down binding buffer, samples were separated by SDS-PAGE, and His-MPK6 was detected by immunoblotting with anti-His antibody. GST alone was used as the negative control. Experiments were performed three times and a representative result is shown. (D) Co-IP assay of AGB1 with AtMPK6. AGB1-Flag and MPK6-Myc or AGB1-Flag and empty pCAMBIA1300 vector (contained Myc-tags) were transiently co-transformed into Arabidopsis protoplast. After 16 h, the total Arabidopsis cell lysates were prepared for Co-IP with anti-Myc agarose. Then, anti-Myc immunoprecipitates were subjected to Western blot analysis with anti-Flag antibody (top). Meanwhile, the total cell lysates were also subjected to Western blot analysis with anti-Flag (middle), and anti-Myc (bottom, for MPK6-Myc expression) antibodies. Experiments were performed three times and a representative result is shown.