Csa001

pDEST27-MRCKα_CAT or pDEST27-MRCKβ_CAT plasmids were transfected in 293T cells with calcium phosphate (Promega). After 36 h, cell lysates were extracted using lysis buffer. GST-tagged protein was isolated through Glutathione Sepharose 4B beads (GE Healthcare). Meanwhile, lysates from MCF10A cells stably infected with the empty vector, PDK1_WT, PDK1_L155E, or PDK1_K465E were extracted using the same lysis buffer. The GST-tagged proteins isolated from 293T bound to glutathione beads were used to pull down proteins from MCF10A extracts. The pulled-down proteins were dissociated using reducing Laemmli buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, and 10% glycerol) and analyzed by immunoblotting.
MRCKα kinase assay was performed by transfecting pDEST27-GST-MRCKα in 293T or HeLa cells overexpressing or silenced for PDK1. After 36 h, cell lysates were extracted using the kinase buffer (25 mM Hepes, 300 mM NaCl, 1 mM PMSF, 1.5 mM MgCl₂, 0.5% Triton X-100, 20 mM Na-β glycerophosphate, 1 mM Na₃VO₄, 0.2 mM EDTA, and 1:1,000 protease inhibitor cocktail; Sigma-Aldrich). Purified GST-MRCKα was assayed for its ability to phosphorylate T696 of recombinant MyPT1 (CSA001; Millipore).