Analysis of the BAFFR interactome and TACI-deficient mice. (A) Left, diagram of the Tnfrsf13c^em1Tyb allele showing exons 1–3 (E1–E3); filled boxes indicate coding sequence; open boxes indicate untranslated regions. Two Strep-tag II sequences were inserted after the last coding codon in E3, before the stop codon. Right, diagram of a BAFFR-Twin-Strep-tag trimer showing the location of the affinity tag at the C terminus of the protein. (B) Volcano plots of proteins identified by mass spectrometry in BAFFR-Twin-Strep-tag APs from B cells activated with LPS, either resting or after stimulation with BAFF for 15 min. The plots show the log₂FC in abundance of each protein in BAFFR APs compared with control APs from WT cells (log₂FC(BAFFR v control)) or in stimulated BAFFR APs compared with resting BAFFR APs (log₂FC(BAFFR +BAFF v BAFFR resting)) from three independent experiments, and the –log₁₀p value was determined by two-tailed Student’s t tests. The thresholds used to determine specific BAFFR interactors (upper right quadrant) are drawn at twofold enrichment and P = 0.05. The full list of proteins identified is in Table S1. (C and D) Numbers (C) and proportions (D) of FO (CD1d^medIgM⁺) and MZ (CD1d^hiIgM^hi) mature splenic B cells (CD93⁻B220⁺CD19⁺) in Tnfrsf13b^+/+ and Tnfrsf13b^−/− mice. Bars show the mean of n = 7 mice, from two independent analyses. Each point represents one mouse. (E) ICOSL surface expression, measured by flow cytometry, on splenic FO B cells (CD1d^medIgM⁺CD93⁻B220⁺CD19⁺) from Tnfrsf13b^+/+ and Tnfrsf13b^−/− mice, quantified as the geometric MFI and normalized to the mean MFI of Tnfrsf13b^+/+ (WT) FO B cells within the same experiment. Bars show the mean of n = 7 mice, from two independent analyses. Each point represents one mouse. Statistical tests: unpaired t test. Numbers above graphs indicate P values where P ≤ 0.05, otherwise no value is shown. MFI, mean fluorescence intensity; FC, fold change; AP, affinity purification.