Question

Paper: TSC2 is Phosphorylated and inhibited by Akt and suppresses mTOR signaling

Insulin Akt Ras12v TSC1-TSC2 TSC1-TSC2 wp-S6 SEK HA-S6K (Th 389) DSGK (Thr421/Ser 424) HA-SEK HA-TSC2 HA-TSC2 Myo-TSC: TSC1-TSC2 SOK (The 389) Insulin (400 M) SOK (Th389) HA-SOK HA-SOK HA-TSC2 HA-TSC2 Myo-TSCH PERK Myc-TSCI PAKT (Ser 473) PAKT (Thr308) GST-ART HA-ERK Figure 1 Inhibition of SOK by TSCI-TSC2. a. TSCI-TSC2 whibits S6K kinase activity. HA-S6K was transfected in HEK293 cells in the presence or absence of TSCI-TSC2, as indicated. HA-SGK was immunoprecipitated and kinase activity was determined using purified GST-S6 as a substrate. The level of HA-S6K in immuno precipitates was determined by immunoblotting with an antisera, Cells were also stimulated with insulin (400 nM) for 30 min or cotransfected with Rasciw, as indicated. b, TSCI-TSC2 selectively inhibits phosphorylation of Thy 389, but not The 421/Ser 424 of S6K. Phosphorylation and protein levels were determined by immunoblotting with appropriate antibodies, as indicated. Cells were also stimulated with insulin, or cotransfected with Rastor GST-Akt, as indicated. c, TSCI-TSC2 does not inhibit Rasinduced activation of p o enotes the probed with the antiphospho ERK antibody, d, Dose dependent inhibition of S6K phosphorylation on The 389 Deft. Akt phosphorylation is detected by phosphospe cific antibodies pakt (Ser 473) and pAkt (Thr 308). TSCI-TSC2 has no effect on either basal or insulin stimulated phosphorylation of Akt (right).

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what were the controls?

what can be concluded and how/why?