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Files in this Data Supplement:
Fig. S1. Expression of endogenous Notch genes and DYRK1A in the cell lines used. (A) RT-PCR analysis of endogenous Notch1-4 in Sh-SY5Y cells. DYRK1A is also expressed in these cells as shown in the right panel. (B) siRNA against DYRK1A is able to reduce the expression of the endogenous gene. C2C12 cells were transfected with the indicated siRNAs and the RNA was extracted and analyzed by RT-PCR. Note the reduction of DYRK1A when compared with the reference housekeeping gene GAPDH.
Fig. S2. Specificity of DYRK-related kinases on Notch signalling. (A) Effect of Ser/Thr kinases on NICD-dependent activation. To determine the specificity of this effect, we used three related proline or arginine-directed protein kinases of the CMGC family. DYRK1B is the closest related member to DYRK1A and their catalytic domains are highly similar, differing only in their amino and carboxy termini. DYRK2 has only 46% identity with DYRK1A, mainly in the catalytic domain, and although CLK-3 belongs to the CLK family, it is more distantly related to DYRK1A. SH-SY5Y cells were transfected with the Nrep reporter and an expression vector for the kinases indicated, in the presence of NICD-S. Relative activity refers to that of the normalized NICD-S alone and the mean error bars represent standard errors from multiple experiments. Despite the differences between the members of the DYRK family, all showed a similar effect to that of DYRK1A. The more distantly related CLK-3, which belongs to the CLK family, had the weakest effect on Notch signalling. (B) Effect of Ser/Thr kinases on the change in NICD mobility. HEK293 cells were transfected with expression vectors for the kinases indicated in the presence of NICD-S. Western blot analysis of the extracts from these transfected cells revealed a shift in the migration of NICD-S associated with members of the DYRK family. Arrowheads indicate the migration of the different proteins.
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