Figure 8.

Phorbol ester sensitivity of Cys2–GFP, Cys1Cys2–GFP and full-length PKC-γ–GFP. (A) In vitro binding of Cys2–GFP, Cys1Cys2–GFP, and PKC-γ–GFP to lipid vesicles in the presence of phorbol ester. In vitro translated ³⁵S labeled fusion proteins were used. The amplitude of each bar represents the percentage of total counts retrieved in the vesicle fraction. The amplitude of each bar represents an average of two samples from the same experiment with the number of counts in the vesicle fraction expressed as a percentage of total counts added (% bound). Two separate experiments with phosphatidylserine vesicles and one experiment with a phosphatidylserine/phosphatidylcholine mixture (1:4 ratio of lipids) gave similar results. (B–D) Series of three images of cells expressing the Cys2–GFP, Cys1Cys2–GFP, and PKC-γ–GFP fusion proteins respectively. The left panels show differential interference contrast images of the cells before stimulation. The middle and right panels show fluorescent confocal fluorescence images recorded immediately before and 5 min after stimulation with 1 μM PMA. All three fusion proteins show maximal translocation of the fusion proteins from cytosol to the plasma membrane in the presence of PMA. For Cys2–GFP and Cys1Cys2–GFP (B and C), the nuclear localized fusion proteins did not significantly redistribute after PMA addition. Images were not corrected for photobleaching.