Figure 5.

Western blot analysis of AANAT and 14-3-3 proteins from FPLC separations. Proteins from nighttime dark-adapted (ZT18), light-treated lactacystin-injected (45 min of light at ZT18, 25 nmol of lactacystin/eye), and light-treated vehicle-injected retinas were separated by FPLC on a Superdex 75 10/300 GL column. Two retinas were used for each separation. Fractions corresponding to the three peaks of AANAT activity were concentrated and subjected to Western blot analysis as described in Materials and Methods. The same blot was labeled with antibodies specific to pan-AANAT (antibody 2992, 1:200), phospho-AANAT (pT²⁹-AANAT; antibody 3352, 1:100), and 14-3-3 proteins (7A3, 1:5000). The approximate molecular weights of the bands are shown. This experiment was repeated twice with similar results.