Fig 2: The MGF_360-4L protein interacts with MDA5. (A and B) HEK293T cells were cotransfected with plasmids expressing Flag-MGF_360-4L and HA-RIG-I, MDA5, MAVS/VISA, TRAF3, IKKε, TBK1, IRF3 or IRF7 as indicated. Co-IP analysis was performed to detect the interaction between MGF_360-4L and RIG-I, MDA5, MAVS/VISA, TRAF3, IKKε, TBK1, IRF3 or IRF7 after 24 h. (C) HeLa cells were transfected with pHA-MGF_360-4L and pFlag-MDA5 or pHA-MGF_360-4L or pFlag-MDA5 with empty vector VEC. The localization of MGF_360-4L and MDA5 was detected via immunofluorescence microscopy. Scale bars = 10 µm. (D) GST-tagged MGF_360-4L protein (10 µg) or GST antigen was added to GST-Sepharose 4B beads, followed by binding with His-tagged MDA5 protein (10 µg). The samples were subjected to western blotting analysis to detect direct interactions between MGF_360-4L-GST and MDA5-His. (E) Schematic diagram of the construction of MGF_360-4L and MDA5 fragments. The long bright blue or grey bands represent the full length, and the colored small bands represent the domains. (F and G) HEK293T cells were cotransfected with Flag-MGF_360-4L (1–3) truncations and pHA-MDA5 (F) or Flag-MDA5 (1–6) truncations and HA-MGF_360-4L (G). Co-IP analysis was performed to detect the interaction between MGF_360-4L and MDA5. (H and I) HEK293T cells were transfected with MGF_360-4L-3 plasmid carrying only the Alpha-lactotoxin_LT1a domain (191–337 aa) with an HA tag and pFlag-MDA5 plasmid, and protein interactions were verified by Co-IP (H). HEK293T cells were transfected with MDA5-5-domain plasmid carrying only the Helicase_C domain (700–839 aa) with a Flag tag and pHA-MGF_360-4L plasmid, and protein interactions were verified by Co-IP (I). (J) HeLa cells were transfected with pFlag-MGF_360-4L-3 and pHA-MDA5 or pFlag-MDA5-5 and pHA-MGF_360-4L. The localizations of MGF_360-4L-3 and MDA5 or MDA5-5 and MGF_360-4L were detected via immunofluorescence microscopy. Scale bars = 10 µm. Fluorescence intensity profiles were measured along the drawn measurement line. (K) HeLa cells were transfected with pHA-MGF_360-4L-3-domian and pFlag-MDA5 or pFlag-MDA5-5-domian and pHA-MGF_360-4L. The localizations of MGF_360-4L-3-domain and MDA5 or MDA5-5-domain and MGF_360-4L were detected via immunofluorescence microscopy. Scale bars = 10 µm. Fluorescence intensity profiles were measured along the drawn measurement line. The representative images of three independent experiments were shown.

Fig 3: Effect of B cell TRAF3 abundance on B cell number and phenotype. (A) Representative western blot of TRAF3 protein in splenic B cells isolated from male and female mice of the indicated genotypes. Each lane is a sample from a single individual. (B) Quantification of splenic B cell TRAF3 protein from western blots including the blots shown in A. (C) Relative Traf3 gene expression in freshly isolated splenic B cells from male and female mice, as determined by qRT-PCR. (D–F) Female mice; (D) Spleen weight at necropsy as a percentage of total body weight in mice of the indicated genotypes. (E) Splenic B cells were quantified after isolation from mice of the indicated genotypes. (F) Germinal Center (GC) B cells quantified by flow cytometry, presented as a percentage of live cells in spleens from mice of the indicated genotypes. (G–I) Male mice; (G) Same as D, for male mice. (H) Same as E, for male mice. (I) Same as F, for male mice. (J) Freshly isolated splenic B cells from male and female mice of the indicated genotypes were cultured in complete medium and counted daily for 7 d to determine survival. (A and B) Data are from two independent experiments with 8 to 9 mice per group. (C) Two independent experiments, 4 to 7 mice per group. (D–I) Three independent experiments, 5 to 14 mice per group. (J) Three independent experiments, 5 to 9 mice per group. Statistical significance was determined by the unpaired t test (B–I) or mixed-effects analysis (J). **P < 0.01, ***P < 0.001.

Fig 4: Impact of decreased B cell TRAF3 on TRAF3-regulated proteins. (A) Representative western blots of Hxk2 (* indicate nonspecific bands), NF-κB2, c-Myc, Mcl1, Pim2, and Actin in splenic B cells from female (Left) and male (Right) mice. Each lane is a sample from a single individual. (B–F) Quantification of the indicated proteins in splenic B cells from female mice, from western blots including those shown in A. (G–K) Same as B–F, for splenic B cells from male mice. Data are from three independent experiments, 6 to 9 mice per group. Statistical significance was determined by the unpaired t test (B–K). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 5: Effect of aging on mouse and human B cell TRAF3. (A) Representative western blot of TRAF3 protein in lysates of B cells isolated from the peripheral blood of healthy donors of the indicated ages. Each lane is a sample from a single individual. (B) Quantification of B cell TRAF3 protein from western blots including the blots shown in A. (C) Relative TRAF3 gene expression in human B cells isolated from the blood of healthy young (17 to 31) or older (60 to 80) donors, determined by qRT-PCR. (D) Representative western blots of Hxk2 (* indicate nonspecific bands), NF-κB2, TRAF3, c-Myc, Mcl1, Pim2, and Actin in lysates of splenic B cells from young (≤3 mos) or older (≥16 mos) female mice. Each lane is a sample from a single individual. (E) Quantification of western blots of B cell TRAF3 protein, including the blots shown in D. (F) Relative Traf3 gene expression in splenic B cells from young and older female mice. (G) Spleen weight at necropsy as a percentage of total body weight in young and older female mice. (H) Number of splenic B cells in young and older female mice. (I) Freshly isolated splenic B cells from young or older female mice were cultured in complete medium and counted daily for 5 d to determine survival. (J–N) Quantification of western blots for the indicated proteins in B cells, including those shown in D. (B) Data are from 10 to 11 individuals per group. (C) Data are from 6 to 10 individuals per group. (D–N) Data are from four independent experiments, 8 to 15 mice per group. Statistical significance was determined by the unpaired t test, **P < 0.01, ***P < 0.001, ****P < 0.0001.