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. 2016 Sep 22;172(3):1973–1988. doi: 10.1104/pp.16.01323

Figure 5.

Figure 5.

The MdBT2-mediated ubiquitination and degradation of MdbHLH104 requires MdCUL3s. A, Immunoblotting assays of MdbHLH104-GFP proteins with the anti-GFP antibody in transgenic calli that had been treated for 6 h with CHX alone or CHX plus MG132 (50 μm). B, Detection of ubiquitination of the MdbHLH104 protein in 35S::MdbHLH104-MYC and 35S::MdbHLH104-MYC+35S::MdBT2 transgenic apple calli pretreated with 50 μm MG132. MdbHLH104-MYC protein was IPed with the anti-MYC antibody. Anti-MYC and anti-ubiquitin antibodies were used to detect MdbHLH104-MYC and ubiquitinated MdbHLH104-MYC, respectively. IB, Immunoblot. C, Pull-down assays with E. coli-expressed His-MdCUL3 result in the precipitation of GST-MdBT2. His was used as the control. Blots were first probed with the anti-His antibody and then stripped and probed with the anti-GST antibody. D, IP assays using the anti-FLAG antibody show the Co-IP of FLAG-MdCUL3 or FLAG with MdBT2 from 35S::FLAG-MdbCUL3+35S::MdBT2-GFP or 35S:: FLAG+35S::MdBT2-GFP transgenic apple calli. E and F, Detection of ubiquitination of the MdbHLH104 protein in the different transgenic apple calli as labeled. The MdbHLH104 protein was IPed with the anti-MYC antibody. The anti-ubiquitin antibody was used to detect ubiquitination of the MdbHLH104 protein.