Fig. 3. Co-immunoprecipitation of p33^ING1b with PCNA. (A) Nondenaturing immunoprecipitations of extracts from 5x10⁶ cells with rabbit polyclonal -ING1 were done in the absence (0) or at the number of hours indicated on the abscissa, after exposure of cells to 25 J/m² of UV. Immunoprecipitates were electrophoresed and blotted with mouse -PCNA. IgG-H and IgG-L represent crossreactivity with heavy and light chains of immunoprecipitating antibodies, respectively. (B) Cells transfected with parental vector pCI^neo (V), or with expression constructs encoding p47^ING1a (a) or p33^ING1b (b) were harvested 24 hours after electroporation under nondenaturing conditions, immunoprecipitated with a mixture of four mouse -ING1 monoclonals (Cab1-4) and electrophoresed and blotted with rabbit -PCNA. (C) Lysates from the same experiment as shown in B blotted with rabbit -PCNA. (D) Lysates from the same experiment as shown in B and C blotted for ING1 expression with Cab1-4. (E) The PIP motif in p33^ING1b where ‘X’ represents any amino acid, ‘h’ represents a hydrophobic residue and ‘a’ represents aromatic residues. ING1b differs from the consensus sequence only by having a valine residue in place of an aromatic amino acid but both valine and phenylalanine have hydrophobic character. (F) Sequence of the PIP box mutations examined in G, showing the relative degree of interaction between PCNA and p33^ING1 variants. Values were generated by scanning densitometry, Results shown are the average of two experiments where data for each was generated by three scans at different exposures. (H) Effects of overexpressing the cyclin-dependent kinase inhibitors p21^WAF1 and p16^MTS1 on the ability of ING1 to bind PCNA. Cells transfected with vector, p21, p16 or p47^ING1a were harvested 48 hours later and immunoprecipitated with -ING1 monoclonals (Cab1-4) under non-denaturing conditions. Immunoprecipitates were electrophoresed and probed with rabbit -PCNA. Experiments were done with both Hs68 and SNB19 cells, and gave similar results.