Receptor occupancy of 14E06 anti-TG2 B cells. (A–C) Splenocytes from Tgm2^+/+ 14E06 KI and HLA-DQ2.5 Tgm2^−/− 14E06 KI mice were prepared and stained for anti-B220, anti-HLA-DQ2.5, and anti-TG2 (14D04). Red blood cells were removed either by ACK lysis or using Lympholyte media. (A) Representative plots gated on B220⁺ splenocytes showing the percentage of B cells with surface-bound TG2. (B) Pooled data of at least 14 Tgm2^+/+ 14E06 KI mice per group showing the percentage of B cells with surface-bound TG2 after red blood cell lysis (ACK lysis) or removal of red blood cells using Lympholyte media. *, P < 0.0001, as determined by Student’s t test. (C) Analysis of mean fluorescent intensity (MFI) of surface anti-TG2 staining on splenic B220⁺ B cells isolated from Tgm2^+/+ 14E06 KI mice. Paired analysis showing representative data from n = 3 mice in one experiment. Data show mean ± SD. *, P < 0.01, as determined by Student’s t test. (D) Flow cytometric detection of phosphorylated proteins in immediately fixed splenocytes isolated from Tgm2^+/+ 14E06 KI (red) and HLA-DQ2.5 Tgm2^−/− 14E06 KI mice (blue) that were processed separately or together. Data are representative of two (D) or three (A and C) independent experiments.