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. 2018 May 14;9:633. doi: 10.3389/fpls.2018.00633

FIGURE 5.

FIGURE 5

Interaction between MtCAS31 and MtLb120-1. (A) Yeast two-hybrid assay to determine the interaction between MtCAS31 and MtLb120-1. MtCAS31 was inserted into pGBKT7 and fused with BD. MtLb120-1 was inserted into pGADT7 and fused with AD. pGBKT7-53/pGADT7-RecT was the positive control. pGBKT7-MtCAS31/pGADT7, pGBKT7/pGADT7-MtLb120-1, and pGBKT7-Lamin/pGADT7-RecT were negative controls. Different co-transformed AH109 yeast cells were dropped on synthetic dropout medium (SD/-Trp/-Leu/-Ade/-His) with 20 mg/mL X-α-gal. (B) In vitro GST pull-down between MtLb120-1 and MtCAS31. Recombinant proteins GST-MtLb120-1 and MtCAS31-His were purified from E. coli. GST-MtLb120-1 was incubated with glutathione beads and incubated with MtCAS31-His. Proteins were eluted from the beads and immunoblotted with anti-His antibody and anti-GST antibody. (C) Interaction between MtCAS31 and MtLb120-1 analyzed by bimolecular fluorescence complementation (BiFC). MtCAS31 was fused with the N-terminus of YFP (MtCAS31-YFPN). MtLb120-1 was fused with the C-terminus of YFP (MtLb120-1-YFPC). Arabidopsis protoplasts were co-transformed with the indicated constructs and incubated for 16 h. MtCAS31-YFPN/MtLb1-YFPC and MtCAS31-YFPN/MtLb2-YFPC were negative controls. Fluorescence signals were observed by confocal laser scanning microscopy with 488 nm excitation. YFP, yellow fluorescent protein; bar = 5 μm. (D) Thermal inactivation assay to determine the protection of MtLb120-1 by MtCAS31. MtLb120-1-GST incubated with or without MtCAS31-His for 4 h. Proteins were heated to 75°C for 45 min for thermal inactivation and then analyzed by native PAGE and immunoblotting with anti-GST and anti-His.