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Figure 9 Determination of PKCa activity after its immunoprecipitation from cells treated with a-tocopherol or p-tocopherol. Cells were stimulated for 7 h with FCS in the absence (Control) or presence of either 50 pM a-tocopherol or (3-tocopherol as indicated. During the last hour of preincubation, they received 100 nM PMA. Then extracts were prepared, PKCa was immunoprecipitated, and a kinase reaction using Histone III-SS was performed as described in Methods. Proteins were resolved by electrophoresis, and radioactive bands were quantified with a BioRad Molecular Analyst software. Protein levels were estimated by staining the gel with the SYPRO kit or by immunoblots with the MC5 monoclonal antibody. The ratio between radioactivity incorporated into the substrate and the amount of PKCa precipitated was expressed as arbitrary units of the densitometric scanning of the bands. Data are representative of three independent experiments.

Figure 9 Determination of PKCa activity after its immunoprecipitation from cells treated with a-tocopherol or p-tocopherol. Cells were stimulated for 7 h with FCS in the absence (Control) or presence of either 50 pM a-tocopherol or (3-tocopherol as indicated. During the last hour of preincubation, they received 100 nM PMA. Then extracts were prepared, PKCa was immunoprecipitated, and a kinase reaction using Histone III-SS was performed as described in Methods. Proteins were resolved by electrophoresis, and radioactive bands were quantified with a BioRad Molecular Analyst software. Protein levels were estimated by staining the gel with the SYPRO kit or by immunoblots with the MC5 monoclonal antibody. The ratio between radioactivity incorporated into the substrate and the amount of PKCa precipitated was expressed as arbitrary units of the densitometric scanning of the bands. Data are representative of three independent experiments.

The inhibition by a-tocopherol of PKCa at a cellular level may thus be related to a possible activation of PP2A by a-tocopherol. This hypothesis was investigated in the experiment outlined in Figure 11. Purified PP2A was incubated with [12P]phosphorylase-a as a substrate; its activation by a-tocopherol, p-tocopherol, and a mixture of the two was analyzed. a-Tocopherol produced almost a twofold activation. (3-Tocopherol was slightly inhibitory, and the mixture of both tocopherols was without significant effect. It thus appears that one of the cellular targets of a-tocopherol may be PP2A.

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