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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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The TRAF3-MFF interaction affected the modifications of MFF and TRAF3. (A, B) Phosphorylation of MFF in resting B cells. Splenic B cells were purified from gender-matched, young adult (8-12-week-old) naïve LMC or B-Traf3 ^-/- mice. Purified cells were analyzed directly (Day 0) or at day 1 after ex vivo culture. S100 (cytosolic), mitochondrial and ER proteins were biochemically fractionated and analyzed by immunoblotting. Proteins in each fraction were immunoblotted for phosphorylated MFF (P-MFF), total MFF, Calreticulin (an ER protein) and COX IV (a mitochondrial protein), followed by TRAF3 and actin. (B) Graphical results of the normalized P-MFF in the mitochondrial fraction. The phosphorylated and total MFF bands on the immunoblots in (A) were quantitated using a low-light imaging system. The intensity of P-MFF bands in each lane of the mitochondrial fraction was normalized to that of the corresponding total MFF bands. Fold of change of the normalized P-MFF relative to that detected for LMC B cells at Day 0 was shown in the graph (mean ± SD, n = 3). **p < 0.01 by t test. (C, D) Ubiquitination of MFF and TRAF3 was affected by the MFF-TRAF3 interaction in 293T cells. Cells were co-transfected with lentiviral expression vectors of HA-tagged ubiquitin (LPA-HA-Ub) and Myc-tagged MFF (pUB-Myc-MFF), SBP-tagged TRAF3 (pUB-TRAF3-SBP-6xHis) or an empty vector pUB-Thy1.1. Transfected cells were harvested at day 2 post transfection. Total cellular proteins were immunoprecipitated with Anti-c-Myc Tag (9E10) Affinity Gel (C) or Streptavidin-Sepharose beads (D). Immunoprecipitates [Myc tag IP in (C) or SA IP in (D)] were immunoblotted for the HA tag, K48-Ub and K63-Ub to detect the ubiquitination, K48- or K63-linked polyubiquitination of MFF (C) and TRAF3 (D), respectively. Phosphorylated MFF (P-MFF) was also analyzed in the Myc tag IP (C). Immunoblotting of Myc-MFF and SBP-TRAF3 was performed with both the immunoprecipitates and input lysates (bottom panels). Immunoblots shown are representative of 3 experiments.