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Generation of diacylglycerol molecular species through the cell cycle: a role for 1-stearoyl, 2-arachidonyl glycerol in the activation of nuclear protein kinase C-ßII at G2/M

¹ MRC Centre for Immune Regulation, Birmingham University, Birmingham B15 2TT, UK
² CRC Institute for Cancer Studies, Birmingham University, Birmingham B15 2TT, UK

View larger version (50K): [in a new window]   Fig. 1. Differential tranlsocation of PKC isoenzymes during cell cycle. U937 cells were synchronised by centrifugal elutriation and proteins were extracted from the cytosol (C) or particulate/membrane (M) fraction of whole cells in G1, S or G2/M. Extracts were analysed by western blotting and probed for the presence of PKC isoenzymes as indicated. The location of an 84 kDa marker protein is shown to the left of the figure. The data shown are representative of four separate experiments.

View larger version (20K): [in a new window]   Fig. 2. Nuclear translocation of PKC-ßII during G2/M. U937 cells were synchronised by centrifugal elutriation. (A) Proteins were extracted from nuclei of unfractionated cells (U) or cells in G1, S or G2/M and analysed by western blotting and probed for the presence of PKC-ßII. Blots were reprobed with an anti-lamin B antibody to confirm equal loading of gels. (B) The densitometric ratio of PKC-:lamin B immunoreactive bands was calculated for cells in G1, S and G2/M. (C) Cells in G1, S or G2/M were indirectly immunostained for PKC-ßII using a FITC-conjugated secondary antibody (green fluorescence) and were counterstained with propidium iodide to locate the nucleus (red fluorescence). Immunofluorescence was visualised using a confocal microscope. The data shown in A and C are representative of three separate experiments, and for B the data are mean±s.d. of three separate experiments; *P<0.02.

View larger version (19K): [in a new window]   Fig. 3. Changes in DAG mass and DAG species in the nucleus during cell cycle. U937 cells were synchronised by centrifugal elutriation and nuclei isolated by a method that retains the double nuclear membrane. Lipids were extracted from isolated nuclei and analysed for (A) total DAG content by DAG mass conversion, expressed as pmoles per 106 nuclei or (B) DAG species by degree of saturation using silver nitrate TLC analysis, expressed as percentage of total nuclear DAG. Data shown are mean±s.e.m. of at least four separate experiments; *P<0.05.

View larger version (19K): [in a new window]   Fig. 4. Activation of classical PKC isoenzymes by SAG in vitro. Recombinant human PKC-, ßII and were combined with SAG in micelle form and PKC activation measured in an in vitro assay by incorporation of [32P]ATP into histone H1 in the presence of calcium and PS. The results are presented as pmol of 32P incorporated per minute per mg PKC and are the mean of triplicates for a single experiment, representative of three separate experiments.