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. 2016 May 3;6:25485. doi: 10.1038/srep25485

Figure 5. Quantitative analysis of IDEF1 and typical iron utilisation-related genes in CSN6 transgenic plants.

Figure 5

(a) IDEF1 protein analysis in CSN6 transgenic rice. Proteins were extracted from 14-day-old WT and T3 generations of CSN6 transgenic seedlings. Immunoblot analysis of native IDEF1 and modified IDEF1 (the high-molecular-weight, HMW) was performed with an anti-IDEF1 antibody in WT and CSN6 transgenic plants (S and A). Equal protein loading was confirmed by immunoblotting with an antibody against HSP80. (b) Western blot analysis of the polyubiquitinated IDEF1 in CSN6 antisense lines. Total proteins of 14-day-old T3 CSN6 antisense lines and WT seedlings were immunoprecipitated by anti-IDEF1 antibody (IP: IDEF1) and detected by an anti-Ub (FK1) antibody (IB: FK1). IB, immunoblotting; IP, immunoprecipitation. Arrows indicate polyubiquitinated IDEF1. Equal protein loading was confirmed by CB (Coomassie Blue stain). Western blot using the anti-IDEF1 antibody was used as a loading control for the input of the IP. (c) qRT-PCR analysis of typical iron utilisation-related genes (IRO2, IRT1, and YSL15) in 14-day-old CSN6 transgenic seedlings. Values represent means with standard deviations (n = 3, P < 0.05 (*), P < 0.01 (**) in Student’s t-test analysis).