Figure 4.

p27 associates with Siah1/SIP in cells. (A) HEK293T cells in 100 mm dishes were transfected with 3 µg each of plasmids producing Myc-tagged p27 and HA-tagged SIP (total DNA = 6 µg). Controls (−) represent cells transfected with HA or Myc-tagged pcDNA3 lacking a cDNA insert. Lysates were prepared and either loaded directly in gels (25 µg total protein content) (labeled as “input”) or used for immunoprecipitations employing anti-Myc antibody. Immune-complexes were analyzed by SDS-PAGE/immunoblotting using anti-HA and Myc antibodies with ECL-based detection. (B) Synchronized wild-type MEFs were cultured in low glucose media and 10% dialyzed FCS and the cytosolic fraction prepared from cell lysates at the indicated times. Endogenous p27 proteins were immunoprecipitated with anti-p27 antibody and immunoprecipitates were analyzed by immunoblotting with anti-Siah1, ubiquitin or p27 antibodies.