rtTA GARP OE mice were given doxycycline to induce GARP expression broadly. (A) Diagram of the experiment scheme. (B) Analysis of GARP and LAP on WT and GARP OE splenic CD19 bead–purified B cells immediately ex vivo (UT) and after 96-hour treatment with anti-μ antibody, LPS, or a combination of anti-μ antibody, CD40L, and LPS. Numbers represent percentage of B220⁺GARP⁺ cells over the gated CD19⁺ B cell population. Flow plots are representative of n = 4 biological replicates. (C) WT and OE splenic CD19⁺ purified B cells were labeled with CFSE and cultured for 3 days with LPS. CFSE dilution was measured by flow cytometry at 24-hour intervals. CD19⁺ purified CFSE-labeled B cells were also cultured with WT non–B cell spleen cells for 72 hours, and CFSE dilution was assessed by flow cytometry. Histograms are representative of 2 independent experiments. (D) Live cell count of 96-hour stimulated cells was analyzed by trypan blue exclusion (n = 4). (E) Total IgA levels in the 96-hour supernatants were measured by ELISA (n = 4). (F) WT and GARP OE mice were given doxycycline in the drinking water for 1 week prior to immunization with TNP-ficoll, and this was continued throughout the experiment. TNP-specific IgM levels were measured in the sera of indicated mice (n = 6 WT and n = 7 OE biological replicates). Statistics were performed by 2-way ANOVA; *P < 0.05. (G) Splenic and Peyer’s patch CD19⁺ bead–purified B cells were isolated from doxycycline-treated WT and GARP OE mice and immediately stained and assessed for intracellular pSmad2/3 levels using flow cytometry (n = 3 biological replicates). Representative of 3 independent experiments. Histograms depict pSmad2/3 staining; gray shaded areas represent the isotype control, black lines represent WT, and green lines represent OE. All statistical analysis was performed by 2-tailed t test unless otherwise indicated; *P < 0.05, **P < 0.01, ***P < 0.001. Error bars represent SD.