Figure 4. GSK3β regulates SCF/SA-mediated mast cell responses independently of mTOR.

HuMCs, transduced with scrambled shRNA (shContr) or shRNA for GSK3β (shGSK3β-B), were sensitized overnight and then stimulated with SA (100 ng/ml) and SCF (30 ng/ml) for 10 min as described in materials and methods. Whole-cell extracts were prepared and immunoblotted with anti-GSK3β anti-p-mTOR(S²⁴⁴⁸), anti-p-p70S6K(T³⁸⁹), anti-p-4E-BP1(T^{37, T46}) antibodies (A). Data in B–E were generated by scanning the blots from 3–5 independent experiments, and normalized to the response obtained at 10 min with SCF/SA stimulation (n=3–5, **p < 0.001 for comparison with SCF/SA response in shContr transduced HuMCs by Student’s t test). In (F), HuMCs were pretreated with Rapamycin (100 nM) or Wortmaninn (100 nM) 20 min prior stimulation with SCF or SA at the time indicated. Whole-cell extracts were prepared and immunoblotted with anti-p-AKT(S⁴⁷³), anti-p-GSK3β(Y²¹⁶), or anti-p-GSK3β(S⁹) antibodies. Protein loading of the samples was normalized by probing for Syk.