Extended Data Fig. 2: Characterization of the Κ-tag system.

(a) Representative flow cytometry plots of peripheral B cells (gated: B220⁺CD4^–CD8^–CD138^–) obtained from the blood of WT and Igk^Tag/Tag mice, stained for surface expression of Igλ and FLAG-tagged immunoglobulins. (b) Total IgG concentration in the serum of WT, Igk^Tag/Tag, and Cd79a^Cre/+. Igk^Tag/Tag mice as determined by ELISA. Differences were not significant by one-way ANOVA (p < 0.05). (c) Flow cytometry of steady-state bone marrow plasma cells (PCs) obtained from adult (6-week old) Igk^FLAG/Strep mice. Gating strategy for PCs is shown in the left panel (pre-gated on non-CD4/CD8 T cells), for surface IgA⁺ and Igλ^– PCs in the middle panel, and for FLAG/Strep in the right panel. Quantification across 9 mice from 3 independent experiments is shown in the rightmost panel (each dot represents an individual Igk^FLAG/Strep mouse, line represents the median). (d) ELISA standard curves with monoclonal antibody (mAb)-FLAG and mAb-Strep detected at dilutions of the respective HRP antibodies where the curves overlap. The mAb concentration at which the curves crossed the absorbance background threshold (indicated by the dotted lines) was used to calculate the endpoint titre, as described in the methods section. (e) Tag-specific anti-TNP titres in Igk^FLAG/Strep mice immunized and boosted i.p. with TNP-KLH/alum at the timepoints indicated by black arrows. Results are from 14 mice from 2 independent experiments. The day 7 timepoint was not collected for the first cohort. Thin lines represent individual mice, thick lines link medians of log transformed titre values at each time point. (f) Flow cytometry of S1pr2-Igk^WT/Tag mice as in Fig. 1d,e. with quantification in (g), cre^– and no tamoxifen control groups are included. Data points are from 6—13 popliteal lymph nodes per group from at least 2 independent experiments. (h) Anti-TNP ELISA reactivity for S1pr2-Igk^Tag/Tag mice immunized as in Fig. 1d. Serum was obtained at 47 d.p.i from 6 mice that received tamoxifen and 4 that did not receive tamoxifen. IgG (left), FLAG (middle) and Strep (right) ELISA absorbance is shown. Samples were diluted 1:100.