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

Figure 2.

Figure 2

MFF is a novel TRAF3-interacting protein. (A) Validation of the functionality of C-terminally SBP-6xHis-tagged TRAF3 in human MM 8226 cells. Reconstitution with the lentiviral expression vector pUB-TRAF3-SBP-6xHis induced apoptosis in 8226 cells as demonstrated by the cell cycle analysis. Cells transduced with an empty lentiviral vector (pUB-Thy1.1) were used as a control. Cell cycle analysis was performed by PI staining and FACS at day 7 post transduction. Gated populations indicate apoptotic cells with DNA fragmentation (DNA content < 2n) and proliferating cells (2n < DNA content ≤ 4n). (B) Large scale affinity purification of SBP-6xHis-tagged TRAF3 from isolated mitochondria of transduced 8226 cells. Cells were harvested at day 4 post transduction and were fractionated to isolate mitochondria. Mitochondrial proteins were immunoprecipitated with Streptavidin (SA)-Sepharose beads. Immunoprecipitates of TRAF3-SBP-6xHis (SA IP) from the mitochondrial proteins were analyzed by immunoblotting and used to identify TRAF3-interacting proteins by LC-MS/MS-based sequencing. Immunoblots of mitochondrial proteins before immunoprecipitation were used as the input control. Cells transduced with FLAG-TRAF3 were used as a negative control for SA IP. (C) Co-immunoprecipitation of MFF and MFF3 with TRAF3. 293T cells were co-transfected with pUB-TRAF3-SBP-6xHis and pcDNA3-Myc-MFF, pcDNA3-Myc-MFF3, pcDNA3-Myc-CSNK2A2 or an empty expression vector pcDNA3-Myc. Transfected cells were harvested at day 2 post transfection and total cellular proteins were immunoprecipitated with Streptavidin-Sepharose beads. Immunoprecipitates (SA IP) were immunoblotted for the Myc tag followed by the SBP tag. Bands of Myc-tagged proteins are indicated. (D) MFF and MFF3 pulled down by GST-TRAF3. Pre-cleared whole cell lysates of 293T cells transfected with pcDNA3-Myc-MFF or pcDNA3-Myc-MFF3 were subjected to the pull-down assay by GST alone or GST-TRAF3 fusion protein in the presence of Glutathione-Sepharose beads. GST and GST-TRAF3 used for pull-down were analyzed by SDS-PAGE and visualized by GelCode blue staining (the bottom panel). Lysates that were purified by the same pull-down procedures with GST were used as a negative control. Myc-tagged MFF and MFF3 in the input lysates and pull-down proteins were detected by immunoblotting (the top panel). Results shown are representative of at least 3 independent experiments.