Figure 6.

V12Rac1 stimulates milk protein synthesis in cells cultured on nonpermissive collagen I substratum. (A) MECs were uninfected or infected with Ad-mycV12Rac1 and plated onto collagen I, where they formed 2D monolayers. Cells were stimulated with Prl for 24 h and were analyzed for β-casein expression by blotting. Infection was confirmed with myc or Rac antibody. These cultures were not overlaid with BM matrix, and it is well established that under these conditions, Prl cannot stimulate differentiation (lane 2). V12Rac1 rescued the defect (duplicate lanes 3 and 4). (B and C) Stat5A nuclear translocation was analyzed in uninfected cells or those infected with Ad-mycV12Rac1, plated onto collagen I–coated coverslips (2D culture with no BM matrix overlay), stimulated with Prl for 15 min, and stained with Stat5a and myc antibodies. (B) Percentage of cells displaying nuclear Stat5a; histogram is a mean of six counts from two experiments. Error bars represent SD. (C) Micrographs demonstrating the nuclear translocation of Stat5a (red) in cells expressing V12Rac1 (green) but not in uninfected controls. Note that monolayer-cultured MECs treated with BM overlay respond to Prl (Fig. 2 C), but in the experiment shown here, no exogenous BM is provided, and the cells cannot respond to Prl (ii). V12Rac1 rescues this defect (iv).