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. 2021 Oct 20;12:670338. doi: 10.3389/fimmu.2021.670338

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Copyright © 2021 Liu, Gokhale, Jung, Zhu, Luo, Saha, Guo, Zhang, Kyin, Zong, White and Xie

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Mapping of the structural domains of TRAF3 required for the interaction with MFF. (A) Schematic diagram of the TRAF3 deletion mutants generated in this study and used for the mapping experiments. Structural domains of TRAF3 that were deleted are depicted in the figure. (B) Interaction with MFF determined by the GST pull-down assay. Pre-cleared whole cell lysates of 293T cells transfected with pcDNA3-Myc-MFF were used in the pull-down experiments by GST alone or GST fusion proteins of WT or deletion mutants of TRAF3 in the presence of Glutathione-Sepharose beads. GST, GST-TRAF3 and GST-TRAF3 deletion mutants used for pull-down were analyzed by SDS-PAGE and visualized by GelCode blue staining (the bottom panel). GST-TRAF3 deletion mutants examined include GST-TRAF3ΔTRAF-C (ΔC), GST-TRAF3ΔTRAF-N&C(ΔN&C), GST-TRAF3ΔZn RING (ΔZnR), and GST-TRAF3ΔZn RING and fingers (ΔZnR&F). Lysates that were purified by the same pull-down procedures with GST alone were used as a negative control and those with GST-TRAF3 were used as the positive control. Myc-tagged MFF in the input lysates and pull-down proteins were detected by immunoblotting (the top panel). Results shown are representative of 3 experiments. (C) Graphical results of the relative association between Myc-MFF and GST fusion proteins of WT or deletion mutants of TRAF3. The Myc-MFF bands on the immunoblots and GST-TRAF3 or mutant bands on the GelCode Blue stained gels in (B) were quantitated using a low-light imaging system and the ImageJ Software, respectively. The amount of Myc-MFF in each pull-down lane was first normalized to the intensity of the corresponding input Myc-MFF band and further normalized to the intensity of the corresponding GST-TRAF3 or mutant band. Fold of change of the normalized Myc-MFF pull-down relative to that detected for GST-TRAF3 WT was shown in the graph (mean ± SD, n = 3). ***p < 0.001 by one-way ANOVA. (D) Direct interaction between TRAF3 and MFF determined by GST pull-down assay of in vitro translated proteins. Pre-cleared protein lysates of in vitro translated Myc-MFF were used in the pull-down experiments by GST alone, GST-TRAF3 or GST-TRAF3ΔC in the presence of Glutathione-Sepharose beads. Myc-tagged MFF in the input lysates and pull-down proteins were detected by immunoblotting (the top panel). GelCode blue staining of GST, GST-TRAF3 and GST-TRAF3ΔC used for pull-down was shown in the bottom panel. Results shown are representative of 3 experiments.