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Figure 5


Fig. 5. A Kalirin antibody specific for Thr1590-P detects Cdk5 phosphorylation of Kalirin-7 and {Delta}Kalirin-7. (A) A 96-well plate was coated with 50 ng nonphosphorylated peptide ({square}) or 50ng QLPK-phospho-T1590PAKLRNNSK ({diamondsuit}). Bound antibody was visualized using alkaline phosphatase-conjugated antibody to rabbit IgG and p-nitrophenylphosphate. (B) The specificity of the Thr1590-P Ab was tested using purified GST-KGEF1-7end and GST-KGEF1-7endT/A. Aliquots of each fusion protein (2 µg) were analyzed directly (0) or after incubation with recombinant Cdk5-p25 complex (2 ng or 10 ng) and 1 mM ATP. Upper panel: western blot analysis with Thr1590-P antibody. Lower panel: Coomassie Brilliant Blue stained membrane. (C) pEAK RAPID cells transfected with vectors encoding Kalirin-7 or parent vector were treated with or without 10 µM roscovitine (Ros) for 4 hours before extraction into SDS lysis buffer. Duplicate samples were visualized using antibody to Thr1590-P (upper panel) or Myc (lower panel). (D) Quantification of Thr1590-P signal normalized to Myc signal.





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