FIG. 5.

Bur1 and Bur2 physical interactions. (A) Coimmunoprecipitation. Extracts were prepared from wild-type haploid strains that express BUR2 in combination with either BUR1 (lanes 1 and 3) or FLAG-tagged BUR1 (lanes 2 and 4). FLAG M2 antibody-conjugated agarose beads were used in immunoprecipitations. Western blot analyses of the crude (lanes 1 and 2) and immunoprecipitated (IP) (lanes 3 and 4) material were performed using anti-FLAG (top) and anti-Bur2 (bottom) antibodies. Fifteen-microgram aliquots of protein were loaded in the crude extract lanes, and the material immunoprecipitated from 250 μg of protein was loaded in lanes 3 and 4. (B) Affinity purification. Extracts were prepared from wild-type haploid strains that express FLAG-BUR1 in combination with either His₆-tagged BUR2 (lanes 1 and 3) or BUR2 (lanes 2 and 4). Proteins were purified using Ni-NTA agarose beads. Western blot analyses of the crude (lanes 1 and 2) and affinity-purified (lanes 3 and 4) material were performed using anti-Bur2 (top) and anti-FLAG (bottom) antibodies. Thirty-microgram aliquots of protein were loaded in the crude extract lanes, and the material affinity purified from 350 μg of protein was loaded in lanes 3 and 4.