Figure 3. TCR activation induces SET-mediated phosphorylation of PP2A_C at Y307 in T_conv cells but not T_reg cells.

(a, b) Naïve CD4⁺ T cells (a) or T_conv and T_reg cells (b) were stimulated with CD3 plus CD28 antibodies for the indicated time periods. Immunoblotting for p-PP2A_C (Y307) was then performed. Data are from one experiment representative of two independent experiments with similar results (n=3 mice per experiment). (c, f) Splenocytes isolated from Foxp3^IRES-GFP mice were stimulated with CD3 plus CD28 antibodies for 24 hours or left unstimulated. Intracellular staining was then performed for PP2A_C (c) or SET (f). Data are from one experiment representative of three independent experiments with similar results (n=3 mice per experiment). (d) Naïve CD4⁺ T cells were stimulated with CD3 plus CD28 for 0, 0.5, 1, 3, 6, 18 and 24 hours. Intracellular staining was then performed for SET and p-PP2A_C (Y307). A representative histogram (left) and quantification of the results (right) is shown from one of two independent experiments with similar results (n=3 per group). (e) Intracellular staining for p-PP2A_C (Y307) of naïve CD4⁺ T cells activated with CD3 plus CD28 antibodies for 24 hours and then spin-infected with an empty or a Set-expressing mCherry lentiviral vector. The analysis was done on mCherry⁺ T cells (n=5 per group, one of two experiments with similar results is shown). Mean ± s.e.m. is shown, MFI: Mean fluorescent intensity, *P<0.01 (unpaired, two-tailed t-test).