FIG. 9.

Photoaffinity labeling of PKCα and PKCɛ by [³H]atRA and specific protection of PKCɛ labeling by 25-hydroxycholesterol. Purified PKCα and PKCɛ were photolabeled with [³H]atRA in the absence and presence of increasing concentrations (0, 5, 25, and 50 μM) of 25-hydroxycholesterol (lanes 3 to 6); lane 2, control with no UV exposure. The photoaffinity labeling was performed as described in the Materials and Methods. The relative photoincorporation of [³H]atRA into the PKC isoforms was determined by densitometry and is shown in the bottom panel. Incorporation in the absence of unlabeled atRA or 25-hydroxycholesterol was assigned a value of 100%. The graph shows the result of quantitation of the protection experiments by densitometry.