Figure 5.

A, Transient expression of indicated hemaggltinin (HA)-tagged and V5-tagged PRLr species in 293T cells was verified by immunoblotting (IB) using indicated antibodies. B, Activity of pyruvate kinase (PK) was determined (as in Fig. 3A) in lysates from 293T cells transfected with vector control (pCDNA3) or vectors for expression of PRLr [wild type or YF mutant (all intracellular tyrosines mutated to phenylalanine, described in Ref. 16)] and treated (100 ng/ml PRL for 20 min, white bars) or not (gray bars) with PRL. Absolute values of PKM activity in untreated cells transfected with either PRLr^WT or PRLr^YF were comparable (data not shown). Average data from four independent experiments (each in triplicate) are presented as percent of activity measured in cells that did not receive PRL (± sd). Asterisks denote statistical significance of obtained differences (P < 0.05 by Student’s t test). C, Lactate levels in the lysates from cells used in panel B were determined as outlined in Fig. 3E. D, PK activity was determined in lysates from 293T cells transfected with vector control (pCDNA3) or vectors for expression of PRLr (wild-type or I170L mutant). Asterisks denote statistical significance of obtained differences (P < 0.05 by Student’s t test). E, Lactate levels in the lysates from cells used in panel D were determined as outlined in Fig. 3E.