Calcium phosphate

293T cells or LR73 cells were transiently transfected with indicated plasmids using calcium phosphate (Promega) or Lipofectamine 2000 (Invitrogen), respectively. After transfection, the cells were lysed and subjected to immunoblotting against the respective tag or proteins. To isolate mitochondria, cells were resuspended in 500 μl of TS buffer (10 mM Tris, pH 7.5, 250 mM sucrose, 2 μg·ml⁻¹ DNase, and protease inhibitor cocktail), and then subjected to three cycles of 5-min freezing in liquid nitrogen followed by 10-min thawing at 37°C. Unbroken cells and nuclei were removed by centrifugation at 800 g for 10 min and mitochondria were collected from the supernatant by centrifugation at 10,000 g for 20 min. The isolated mitochondria were lysed in RIPA buffer and subjected to immunoblotting. To stain cells for microscopy, LR73 cells were plated and transfected with FLAG-tagged Ucp2. One day after transfection, the cells were incubated with Mitotracker Deep Red (100 nM, Molecular Probes) in the culture medium for 20 min. The cells were then fixed with 3% paraformaldehyde (Sigma) in PBS for 30 min, permeabilized with 0.1% Triton X-100 (Sigma) and blocked with 5% clarified milk. Antibody staining was then performed using antibodies to FLAG (Clone M5, Sigma) and AIF (D39D2, Cell Signaling Technologies). The stained cells were analyzed by Axio Imager D2 (Zeiss).

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).