Figure 8

Primary mammary epithelial cell cultures confirm that Src is required for maximal prolactin signaling. Primary mammary epithelial cells isolated from Src-/- (lanes 1–3 and 10–12), Src+/- (lanes 4–6 and 13–15) and Src+/+ (lanes 7–9 and 16–18) mice were cultured on Matrigel in growth medium for 3 days then the medium was changed to differentiation medium and the cells were cultured for a further 24 or 72 hours in the presence or absence of prolactin. Cellular proteins were extracted by lysis in NET buffer. A) Immunoblotting was performed to detect phosphorylation of Src (tyrosine⁴¹⁶) (top panel), total levels of Src protein (middle panel) and the amount of ERK 1&2 (bottom panel) as a loading control. B) Immunoblotting was performed to detect phosphorylation of STAT5 (tyrosine⁶⁹⁴) (top panel), total levels of STAT5 (middle panel) and the amount of ERK 1&2 (bottom panel) as a loading control. C) Immunoblotting was performed to detect β-casein expression (top panel), the levels of Raf and the amount of ERK 1&2 as loading controls (middle and bottom panels).