FIG. 1.

(A) Thirty-five-millimeter-diameter dishes of COS1 cells were transfected with 0.5 μg each of expression vectors for HA-Cul3 and Flag-CAND1 as indicated. Total cell lysates were analyzed by immunoblotting with anti-Flag (α-Flag) and α-HA antibodies (bottom two panels). α-Flag immunoprecipitates (IP) were subjected to immunoblot analysis using α-HA antibodies (top panel). α-HA IP were subjected to immunoblot analysis using α-Flag antibodies (second panel from the top). (B) Thirty-five-millimeter-diameter dishes of COS1 cells were transfected with 0.33 μg each of expression vectors for Keap1-CBD and HA-Cul3 as indicated. The Flag-CAND1 expression vector was either omitted (lanes 1 to 3) or included (lane 4). Total cell lysates were analyzed by immunoblotting with α-CBD, α-Flag, and α-HA antibodies (bottom three panels). The lysates were incubated with chitin beads, pelleted by centrifugation (3,000 × g), and washed three times in lysis buffer. Proteins that remained associated with the chitin beads were analyzed by immunoblotting with α-HA antibodies (top panel). (C) Thirty-five-millimeter-diameter dishes of COS1 cells were transfected with expression vectors for HA-Cul3 (lanes 1 to 5) and GAN-CBD (lanes 2 and 3) or sarcosin-CBD (lanes 4 and 5). The Flag-CAND1 expression vector was either omitted (lanes 1, 2, and 4) or included (lanes 3 and 5). Cell lysates were analyzed by immunoblotting with the indicated antibodies (bottom three panels) or incubated with chitin beads. Proteins that remained associated with the chitin beads after extensive washing were analyzed by immunoblotting with α-HA antibodies (top panel).