Supplementary Figure 3: T_reg cells have decreased phosphorylation of PP2Ac at Tyr307 upon activation through modulation of the ceramide-SET-PP2A pathway.

(a) Splenocytes isolated from Foxp3^IRES-GFP mice were stimulated with CD3 plus CD28 (2μg/mL) antibodies for 24 hours. Intracellular staining was then performed for p-PP2A_C (Y307) in CD4⁺Foxp3^- and CD4⁺Foxp3⁺ T cells (n=3 per group). Data are from one experiment representative of three independent experiments with similar results. (b) Intracellular staining for p-PP2A_C (Y307) of naïve CD4⁺ T cells transfected with a scramble shRNA or Set-specific shRNA mCherry-expressing plasmid and then activated with CD3 plus CD28 (2μg/mL) antibodies for 24 hours. The analysis was done on mCherry⁺ T cells (n=3 per group). Data are from one experiment representative of two independent experiments with similar results. (c) Jurkat T cells were treated with SMase or vehicle for 1h and then lysed for protein immunoprecipitation with a PP2A_C-specific antibody or a mouse IgG control antibody. The blots were then probed for SET and PP2A_C. Image is from one experiment representative of two independent experiments with similar results. (d) Naïve CD4⁺ T cells were stimulated with CD3 plus CD28 (2μg/mL) antibodies for 24h and then treated with vehicle or SMase for 1 or 2 hours. The cells were then lysed for immunoblot and probed for p-PP2A_C (Y307) and total PP2A_C. The relative ratio of p-PP2A_C/total PP2A_C band densities for each lane is also shown. Image is from one experiment representative of two independent experiments with similar results. Mean ± s.e.m., MFI: Mean fluorescent intensity, *P<0.05, **P<0.01 (unpaired, two-tailed t-test).