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Fig. 3. SHH signaling inhibits S6 kinase activation and hence IRS1
degradation. (A) Treatment of CGNPs with 1 µg/ml cyclopamine
after increasing periods of time reduced IRS1 protein levels and increased
detectable phosphorylated IRS1. The value under each lane represents the ratio
of phosphorylated IRS1 to IRS1 as measured by densitometry. (B) CGNPs
were treated with SHH and/or 10 nm rapamycin (Rapa) as indicated. The final
lane represents CGNPs from which SHH was removed at the time of rapamycin
addition. Rapamycin in combination with SHH causes an accumulation of IRS1 and
rapamycin prevents reduction in IRS1 when SHH is removed. The value under each
lane represents densitometric measurement of the IRS1 signal using the
vehicle:tubulin value set to 1. (C) Treatment of CGNPs with rapamycin
in the presence of SHH caused an accumulation of IRS1 over time. The value
under each lane represents densitometric measurement of the IRS1 signal using
the vehicle:tubulin value set to 1. (D) SHH, cyclopamine and rapamycin
were given to CGNPs, as indicated above the lanes. Cyclopamine caused
reduction in IRS1 levels, whereas rapamycin caused additional IRS1
accumulation in the presence of SHH. Rapamycin partially rescued the
cyclopamine-mediated reduction of IRS1 in SHH-treated cells. (E)
Treatment with SHH reduces the phosphorylation of S6K and hence its
activation. Inhibition of PP2A with okadaic acid (OA, 100 nM) restores S6K
activation and reduces N-myc as well as cyclin D2 levels in CGNPs. (F)
Treatment with OA restores S6 phosphorylation and reduces IRS1 levels even in
the presence of SHH.
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