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. Author manuscript; available in PMC: 2013 Aug 1.
Published in final edited form as: J Immunol. 2013 Mar 25;190(9):4508–4515. doi: 10.4049/jimmunol.1202923

FIGURE 3.

FIGURE 3

PDK1 peptides carrying Thr-513 to Ile and Asp mutations binds to PDK1 kinase domain and PH domain, respectively. A, Sequence alignment between PDK1 target phosphorylation sites and the PDK1 Thr-513 region was performed in ClustalW. B, PTD-PDK1-Thr-513-Asp and PTD-PDK1-Thr-513-Ile were designed as indicated. PTD peptide was used as a control. C and D, Chemically activated M450 beads were conjugated with PTD peptide, PTD-PDK1-Thr-513-Ile, or PTD-PDK1-Thr-513-Asp. The conjugated peptides were incubated with lysate from HEK293 cells transfected with PDK1 kinase or PDK1 PH domain expression plasmids (C) or incubated with lysate from mouse primary CD4+ T cells stimulated with anti-CD3 and anti-CD28 antibodies for 1 hour (D). Binding proteins were analyzed by immunoblotting (C and D).