Immunodepletion studies of cyclin D1 and p27^Kip1.

(A) The cell lines Granta 519 (G), NCEB-1 (N), and KMS12 (K) are sequentially immunoprecipitated with anticyclin D1. By the third immunoprecipitation (3rd IP) all 3 cell lines were depleted of cyclin D1. (B) Supernatants were immunoprecipitated with p27^Kip1; however, once cyclin D1 was depleted (4th IP), p27^Kip1 was not detectable. (C) In the inverse assay, the 3 cell lines were sequentially immunoprecipitated with anti-p27^Kip1. p27^Kip1 was depleted by the second immunoprecipitation. (The bands observed in the blot are nonspecific bands seen with the polyclonal p27^Kip1 antibody.) (D) The remaining supernatants were immunoprecipitated with anticyclin D1 (3rd IP). The recovery of cyclin D1 in the absence of p27^Kip1 reveals excess of cyclin D1 relative to p27^Kip1. C indicates positive control; IP, immunoprecipitation; Sup, supernatant.