Figure 2.

SFB4′ protein does not interact with S₄-RNase. A, Pull-down assay to detect the interaction between S₄-RNase and SFB4/SFB4′. SFB4 and SFB4′ were fused with SUMO and His tags, and the molecular masses were almost 55 and 45 kD, respectively. S₄-RNase was fused with an MBP tag, and the molecular mass reached around 72 kD. SFB4 and SFB4′ were detected by His tag monoclonal antibody, while S₄-RNase and MBP were detected by MBP monoclonal antibody. B, BiFC assay to detect the interaction between S₄-RNase and SFB4/SFB4′. S₄-RNase was fused with the N-terminal end of YFP, SFB4 and SFB4′ were fused with the C-terminal end of YFP, and the combinations S₄-RNase/SFB4, S₄-RNase/SFB4′, S₄-RNase/YFPc, and YFPn/SFB4 were expressed in N. benthamiana leaves. DIC, Differential interference contrast. Bars = 20 μm. C, Semi-in vivo pull-down assay to further detect the interaction between S₄-RNase and SFB4/SFB4′. Pollen tube proteins from cv Bin and cv 21-21 were incubated with MBP protein and MBP-tagged S₄-RNase. The molecular masses of SFB4 and SFB4′ were almost 45 and 39 kD in pollen tubes, respectively. D, Semi-in vivo immunoprecipitation (IP) assay to confirm the interaction. Pistil proteins from cv 21-21 were incubated with pollen tube proteins from cv Bin and cv 21-21. The molecular mass of S₄-RNase was almost 30 kD. The asterisk represents the signal of S₄-RNase. The higher molecular mass band is a nonspecific band. α-His, His tag antibody; α-MBP, MBP tag antibody; α-S₄-RNase, S₄-RNase antibody; α-SFB4, SFB4 antibody.