We have tried IP for total protein with detection using p-Tyr antibody, but we can only occasionally detect a signal. Are the proportion of PDK1 that gets tyrosine-phosphorylated extremely small, meaning that you have to use cells that overexpress PDK1, or..!? There are WB antibodies that detect PDK1 only when it is phosphorylated at Tyr9, but none that specificallty detects PDK only when is phosphorylated at Tyr373/Tyr376...
There has been a paper in JBC by Park et al quite a while ago (Vol. 276, No. 40, Issue of October 5, pp. 37459–37471, 2001) where site spec pTyr Antibodies against Y9 and Y373/376 were used. Brian Hemmings is still at the FMI in Basel, perhaps there are aliquots of the antibodies available.
Hi
Is not PKD1 phosphorylated by Src? Thus could you try to see PKD1 phosphorylation in Src transformed cells?
Best
Daniel
- Badges
- Science method