Figure 2.

Rb/p130 Interaction with CtIP. (A) Schematic representation of the CtIP protein. The position of an LXCXE sequence motif and a PLDLS sequence motif within the 897-aa CtIP protein are indicated. (B) HF7C yeast were transformed with plasmid encoding the Gal4AD-CtIP fusion protein alone or with a Gal4BD-p130, Gal4BD-Rb, Gal4BD-KSS1, or the empty Gal4BD vector. Yeast were streaked on nonselective media lacking Trp and Leu and on media that lacks Trp, Leu, and His that is selective for protein/protein interactions. (C) C33A cells were transfected with 10 μg of Myc-CtIP and 10 μg of either Gal4-p130 or Gal4-p130^C894F. Cells were harvested 40 hours posttransfection and were lysed in IP buffer. Ten percent of the extract was loaded in input lanes 1–3. Myc antibody (Santa Cruz Biotechnology, 9E10) was used to immunoprecipitate Myc-CtIP. p130 antibody (Santa Cruz Biotechnology) was used in Western blotting to detect p130 in the immunoprecipitates (lanes 4–6). The blot was stripped and reprobed with Myc antibody to verify that equal amounts of Myc-CtIP were immunoprecipitated (lanes 4–6). (D) C33A cells were transfected as in C with 10 μg of Gal4-p130 and 10 μg of either Myc-CtIP or Myc-CtIP ΔLXCXE. Ten percent of the extract was loaded in input lanes 1 and 2. Myc antibody was used to immunoprecipitate Myc-CtIP. p130 antibody was used in Western blotting to detect p130 in the immunoprecipitates (lanes 3 and 4). The blot was stripped and reprobed with Myc antibody to verify that equal amounts of Myc-CtIP were immunoprecipitated (lanes 3 and 4).