Figure 2
Characterisation of the T-cell infiltrate. A, B, C: Cell suspensions from transgenic St3/4 (Tg) and control (NSC) ear tissue (approximately 106 cells/sample, each from two transgenic ears or 4 to 6 control ears) were analysed by flow cytometry using fluorescent-conjugated antibodies. In all cases, the leukocyte populations were first gated by forward and side scatter as shown in the first panel of A. Viable leukocytes were further gated as the CD45+(APC)/7-AAD-(PerCP) population shown in the middle panel of A. Cells were further stained using antibodies directed to CD4(PE) and CD8(FITC) (A), NK1.1(PE) and CD3(FITC) (B) and CD8(FITC) and Granzyme B(PE) (C); showing proportions of cells in relevant quadrants or gates. CD4/CD8 proportions in (A) are given as the % of CD45+/7-AAD- cells (black) and as the % of the gated FSC/SSC cells (red). D: Protein was extracted from control (NSC: C5) and transgenic ears (St5). Three biological replicates (40 μg) were western blotted and probed with anti-TGFβ1 (upper panel: 15% gel, precurser dimer: 44kD, mature protein dimer: 25kD, under these reducing conditions: 22 and 12.5kD respectively) or α-Rae1 (lower panel: 12.5% gel, migrates at ~40kD). α-GAPDH was used as a loading control as indicated (37kD).