Differentiation-dependent protein expression of PRL and IGFBP1 in culture supernatants of HDSC and THESC. Cells were stimulated in the absence or presence of cAMP and/or E2P4 for 3, 6, 9 and 12 days. Supernatants were collected and PRL and IGFBP1 protein concentrations were measured by ELISA and Western blotting, respectively. Normalization of protein concentrations, ELISA, and immunodetection of IGFBP1 on membranes were performed as mentioned in Materials and Methods. A) ELISA of PRL in HDSC and THESC. Data represent mean values ± SEM of three experiments performed in duplicates. * indicates p < 0.05 compared to negative control (n.c.) of the same day; n.s., not significant; # indicates p < 0.05 between cAMP-treated and cAMP/E2P4-stimulated cultures. PRL expression in controls and E2P4-treated THESC were below the detection limit and therefore omitted from the graph. B) Western blot showing IGFBP1 (32 kD) expression in HDSC and THESC. Marker bands (kD) are depicted on the left side. Representative examples are shown.