In vivo association between DNMT1 and PKCζ. (A) Co-immunoprecipitation of DNMT1 and PKCζ in nuclear extracts of HEK-293 cells. The cells were transfected with DNMT1 and PKCζ-c-myc or c-myc for 48 hours and c-myc proteins were purified with immobilized anti-c-myc beads. Protein complexes were resolved by SDS/PAGE and the presence of PKCζ was demonstrated using an anti-c-myc antibody; DNMT1 and actin were revealed, respectively, using an anti-DNMT1 and an anti-actin antibody. (B) Detection of endogenous PKCζ activity in DNMT1 immunoprecipitates. Nuclear proteins from HEK-293 cells were incubated with beads prebound to an isotopic IgG antibody or antibodies against DNMT1 or PKCζ for 4 hours. After several washes, protein-bead complexes were tested for kinase activity using (γ-32P)ATP and PKCζ specific substrate. Data are representative of three independent experiments. rec. PKCζ, recombinant PKCζ.