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

Figure 6.

Figure 6.

MdBT protein accumulation is dependent on iron. A, Immunoblot analysis of MdBT1-GFP and MdBT2-GFP proteins with the anti-GFP antibody under iron-deplete or iron-replete conditions. The 35S::MdBT1-GFP and 35S::MdBT2-GFP transgenic apple calli were used. B and C, Time course of changes in the abundance of the MdBT2 and MdbHLH104 proteins in response to iron availability. Transgenic apple calli were incubated with (+Fe) or without (−Fe+Frz) iron for 1 d, and then switched to no (−Fe+Frz) iron or iron (+Fe) conditions, respectively. D, The 35S::MdbHLH104-MYC and 35S::MdbHLH104-MYC+35S::MdBT1/2-anti transgenic apple calli were treated with (+Fe) or without (−Fe+Frz) iron and IPed with the anti-MYC antibody. Ubiquitin conjugates that were immunoprecipitated were detected with anti-ubiquitin and anti-MYC antibodies, respectively. IP, Immunoprecipitated; IB, immunoblotted. E, Fe-oversupply-mediated degradation of the MdbHLH104 protein is dependent on MdBT1 and MdBT2. 35S::MdbHLH104-MYC transgenic apple calli were treated without iron (−Fe+Frz) and then given iron (+Fe) in the presence or absence of MG132 (50 μm). The 35S::MdbHLH104-MYC+35S::MdBT1/2-anti transgenic apple calli were given iron (+Fe). Total protein extracts were used for immunoblotting with the anti-MYC antibody. ACTIN was used as the loading control.