FIG. 7.

Metabolic labeling experiments. (A) Pulse-labeling of MCF-7 cells. Serum-starved cells were pretreated with no drug, heregulin, or heregulin plus rapamycin for 30 min in Met-free medium. [³⁵S]Met-Cys was added, and the cells were incubated for 20 or 40 min prior to preparation of cell lysates and immunoprecipitation of HIF-1α. (B) Pulse-chase. Serum-starved MCF-7 cells were pretreated with 100 μM cobalt chloride (C) or 100 ng of heregulin/ml (H) for 30 min in Met-free medium. [³⁵S]Met-Cys was added, and then the cells were incubated for 40 min and either harvested directly for analysis of pulse-labeling (P) or rinsed and incubated in medium containing unlabeled Met-Cys for 25 min in the presence or absence of heregulin for pulse-chase analysis (P/C) of immunoprecipitated HIF-1α.