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. 2017 Feb 8;7:42123. doi: 10.1038/srep42123

Figure 4. Specificity determinants for the interaction between TOP3β and TDRD3.

Figure 4

(a) Amino-acid sequences of the insertion loop region in the TDRD3 mutant containing the RMI1 insertion loop (TDRD3-RMI1loop), the A80S/A81Q/A90K/A91P/P92S mutant of TDRD3 and the RMI1 mutant containing the TDRD3 insertion loop (RMI1-TDRD3loop). (b) GST pull-down assays of TDRD3-RMI1loop, the A80S/A81Q/A90K/A91P/P92S mutant and RMI1-TDRD3loop. The GST-TOP3β-bound resins were incubated with 1.0 μM wild-type or mutant proteins of TDRD3 or RMI1. Shown are the cropped gel images. The contrast was adjusted for clarity. Quantitative analyses and original full-length gel images corresponding to the left and right panels are shown in Supplementary Fig. 7a. (c) GST pull-down assays of TDRD3-RMI1oop at different concentrations (0.01–0.5 μM). TDRD3-RMI1loop could bind TOP3β, but with lower affinity than wild-type TDRD3. Shown are the cropped gel images. The contrast was adjusted for clarity. Quantitative analyses and original full-length gel images corresponding to this figure are shown in Supplementary Fig. 7b. (d) GST pull-down assays of the TDRD3 mutants containing R96M, V109M, F111Y, F139V or their combinations and the M149V/Y151F/V179F mutant of RMI1. The GST-TOP3β-bound resins were incubated with 0.5 μM wild-type or mutant proteins of TDRD3 or RMI1. Shown are the cropped gel images. The contrast was adjusted for clarity. Quantitative analyses and original full-length gel images corresponding to this figure are shown in Supplementary Fig. 7c. (e) GST pull-down assay of the TDRD3-RMI1oop mutant containing the R96M, V109M and F139V mutations. The GST-TOP3β-bound resins were incubated with 0.5 μM wild-type or mutant proteins of TDRD3 or RMI1. Shown are the cropped gel images. Quantitative analyses and original full-length gel images corresponding to the left and right panels are shown in Supplementary Fig. 7d.