Fig. 7: p15, but not p16, binds enolase-1 in vitro and in vivo.

a (left panel) Protein pull-down (PD) using E. coli-expressed, histidine-tagged and nickel column-immobilized p15 or p16 in the presence of total proteins from parental UMUC3 cells, and (a, right panel) immunoprecipitation (IP) of Myc-tagged p15 or p16, followed by western blotting. Note in both experiments the highly specific interaction of p15, but not p16, with enolase-1 with a molecular weight of 48-kDa. The side bars in the top two western blotting strips in the right panel denote molecular weight standards, 55-kDa (upper bar) and 43-kDa (lower bar). b p15 specifically interacts with enolase-1 in vivo. Total proteins were extracted from mouse urothelial cells expressing HRas* or those expressing HRas* and lacking p15, and were then subjected to immunoprecipitation followed by western blotting. One mouse per lane representative of a total of three independent mice per genotype. Note that enolase-1 was immunoprecipitated by anti-p15 antibody in p15-expressing, but not in p15-lacking urothelial cells (despite the fact that enolase-1 was significantly elevated in these cells); that p15 did not immune-precipitate AKT or PKM2; and that p16 did not immuno-precipitate enolase-1. c p15 binds, via its N terminus, to enolase-1. Myc-tagged, domain-switching mutants of p15 and p16 and wild-type controls were stably transfected and inducibly expressed in parental UMUC3 cells, and anti-Myc immunoprecipitation followed by western blotting was carried out. Note the specific immunoprecipitation of enolase-1, but not enolase-2, by p15 and p15-N/p16C, but not by p16 or p16-N/p15-C. Experiments in a–c were repeated three times with similar results. Source data are provided as a Source Data file.