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. 2012 Dec 10;288(4):2829–2838. doi: 10.1074/jbc.M112.404095

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FIGURE 5. — Thr(P)-383 phosphorylation site-specific antibody recognizes PKCϵ-phosphorylated but not nonphosphorylated or dephosphorylated hTASK-1 C terminus fused to GST. Western blot analysis of in vitro kinase reactions of WT or Thr-383A hTASK-1 C terminus fused to GST that were phosphorylated (1st and 4th lanes) or not phosphorylated (2nd lane) by PKCϵ (left panel). Some reactions were subsequently dephosphorylated by acid phosphatase (5th lane, right panel). Proteins were resolved by SDS-PAGE and transferred to nitrocellulose. Phosphorylated Thr-383 was detected by immunoreactivity with a phosphorylation site-specific antibody (top panel), and total protein loading was confirmed by immunoreactivity with an anti-GST antibody (bottom panel). The Thr(P)-383 antibody is specific for the phosphorylated C terminus (1st and 4th lanes), and it does not recognize protein that was not exposed to PKCϵ (2nd lane), protein where the Thr-383 phosphorylation site was mutated (3rd lane), or protein that was dephosphorylated (5th lane). IB, immunoblot.