Journal: The Journal of Biological Chemistry

Article Title: The Role of Regulatory Domains in Maintaining Autoinhibition in the Multidomain Kinase PKCα *

doi: 10.1074/jbc.M116.768457

Figure Lengend Snippet: Disrupting PKCα autoinhibition increases membrane translocation. A, upon stimulation with 0.05 mg/ml of DiC8 lipid, HeLa cells were imaged for 10 min at 22 °C using a confocal microscope. Movies were analyzed by selecting and monitoring fluorescence cytosolic intensity over time for three cytosolic regions per cell. B, representative images and C, analysis of one field of cells per condition (mean ± S.E., n ≥ 2 cells). For each condition, at least two independent experiments were performed and in total more than four cells per condition were analyzed. Autoinhibition was increasing disrupted by introducing either 10- or 30-nm ER/K linkers between the catalytic domain and regulatory domains, as previously described (20). The rate of translocation was compared with mCitrine-labeled full-length PKCα and a mCitrine (mCit) fluorophore control.

Article Snippet: Once a region was identified with at least two suitable cells, protein localization was then monitored for 10 min upon addition of 1 ml of HBS buffer containing 2 m m CaCl 2 and either 0.1 mg/ml of 1,2-dioctanoyl- sn -glycerol (DiC 8 ; Avanti) ( 26 ) or 2 μ m phorbol 12-myristate 13-acetate (EMD) ( 20 ).

Techniques: Translocation Assay, Microscopy, Fluorescence, Labeling