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. 2020 May 1;295(24):8236–8251. doi: 10.1074/jbc.RA120.012572

Figure 8.

Figure 8.

TGF-β receptor signaling promotes P85 stabilization of PTEN and AKT activation. Simulations were performed using a constant amount of TCR signal and varying amounts of TGFβR signal. A, trajectories for total PTEN abundance is presented. B, CD4+ T cells isolated by negative selection were in vitro activated through TCR and CD28 costimulation for 10 min with various doses of TGF-β. Immunoblotting was performed for total PTEN and actin. C, densitometry was performed from three independent experiments where PTEN abundance was normalized to actin. Shown are mean ± S.D.; p values were calculated by one-way ANOVA with a Tukey multiple comparison test. D, CD4+ T cells isolated by negative selection were in vitro activated through TCR and CD28 costimulation for 10 min with ± 10 ng/ml TGF-β. P85 was immunoprecipitated from cell lysates. Immunoblotting was performed for PTEN and P85. One representative blot from three independent experiments is shown. E, densitometry across three independent immunoblots was performed for PTEN normalized to total P85 in the IP. Shown are mean ± S.D.; p values were calculated by a Student's t test. Simulations were performed using a constant amount of TCR signal and varying amounts of TGFβR signal. F, trajectories for p-AKT (Ser-473) abundance are presented. Murine CD4+ T cells isolated by negative selection were in vitro activated through TCR and CD28 costimulation for various time points with or without 10 ng/ml TGF-β. G, immunoblotting was performed on cell lysates for the phosphorylated and total protein abundance for phosphorylated AKT (Ser-473). H, densitometry was performed across three independent experiments to quantitate the abundance of phosphorylation for AKT (Ser-473). The abundance of phosphorylated protein was normalized to total protein. Shown are mean ± S.D.; p values were calculated by two-way ANOVA. I, CD4+ T cells were activated in the presence of different doses of TGF-β and immunoblotting was performed for p-AKT (Thr-308), p-AKT (Ser-473), and total AKT. J and K, densitometry across three independent immunoblots was performed for (J) p-AKT (Thr-308) normalized to total AKT and (K) p-AKT (Ser-473) normalized to total AKT. Shown are mean ± S.D.; p values were calculated by one-way ANOVA with a Tukey multiple comparison test.