FIG. 4.
In vitro dimerization activity of the mutants. 35S-labeled, Flag epitope-tagged wt PKR and DRBD proteins were in vitro translated in rabbit reticulocyte lysate. Equal amounts of the two proteins to be tested for interaction were mixed and incubated at 30°C for 30 min with anti-Flag MAb-agarose in 50 μl of immunoprecipitation buffer. The proteins remaining bound to agarose beads after extensive washing were analyzed by SDS-PAGE followed by fluorography. Lanes 1 and 7, Flag-tagged wt PKR and wt DRBD; lanes 2 and 8, Flag-tagged wt PKR and A63E DRBD; lanes 3 and 9, Flag-tagged wt PKR and A67E DRBD; lanes 4 and 10, Flag-tagged wt PKR and L75E DRBD; lanes 5 and 11, Flag-tagged wt PKR alone; lane 6 and 12, wt DRBD alone. The positions of PKR and DRBD are indicated by arrows. Lanes 1 to 6 show total proteins from reticulocyte lysate, and lanes 7 to 12 show immunoprecipitated proteins.