Fig 1: Confirmation of proteomic data by flow cytometry and Western Blotting. (A–C) Mouse bone marrow cells were stained with antibodies against CD19, B220, CD138, TACI, fixed, permeabilized, and stained with rabbit anti JCHAIN antibody followed by a secondary antirabbit antibody. (A) Plasma cell gate (TACI+, CD138+) and (B) B cell gate (CD19+, B220+). (C) JCHAIN expression in B cells or plasma cells: mean fluorescence intensity (MFI) is depicted below. (D) CH12tfgKO and WT cells were stained with antirabbit antibody alone or with rabbit anti JCHAIN antibody followed by a secondary antibody. MFIs are depicted below. (E) Quantification of JCHAIN abundance in CH12tfgKO and WT cells after background subtraction of secondary antibody staining. Two experiments (experiments) with five different clones per genotype in total are shown. Each dot represents one clone. Mean of median FI ± standard deviation (SD). Statistical significance (experiment 1) was calculated by Student’s t-test. (F) CH12tfgWT and KO B cells (7 clones each) were stained with antibodies against surface CD20 and CD98 or fixed, permeabilized, and stained with antibodies against CD74 and then analyzed by flow cytometry. The fluorescence intensity was normalized to the cell size and is depicted as the median intensity. Each dot represents one clone with mean ± SD. Statistical significance was calculated by the Mann–Whitney U-test. (G) Fold-change of protein abundance of CH12tfgKO B cells was calculated relative to CH12tfgWT cells for flow cytometry and correlated to the relative changes as quantified by LC-MS/MS. (H) CH12tfgKO and WT B cells were lysed and subjected to Western Blotting with antibodies as indicated, and a part of the blot is shown. (I) Quantification of the Western Blot shown in panel (G). The background was subtracted and the optical density (OD) of the given protein was normalized to the OD of Actin. Statistical significance was calculated by Mann–Whitney U-test.