Fig. 2. Cytoskeletal inhibitors affect ß-casein expression in primary mammary epithelial cells First-passage mammary epithelial cells were plated on basement membrane in DMEM-F12 supplemented with insulin and hydrocortisone. Cytoskeletal inhibitors and prolactin were added for the indicated times and cells were extracted for protein and RNA analysis. (A-C) Cells were ³⁵S-methionine labelled for 1 hour. Equal amounts of trichloroacetic-acid-precipitable counts were either directly analysed by gel electrophoresis to detect newly synthesized proteins (A) or immunoprecipitated with rabbit anti-mouse milk antiserum before SDS-PAGE, for ß-casein detection (B,C). Notice that the overall spectrums of newly synthesized proteins in (A) are not significantly affected by drug treatment. The asterisk in (A) corresponds to ß-casein, the levels of which are sufficient to be visualized within the total cell proteins 24 hours after inducing differentiation. Notice also that the exposure time for the gel in (C) is considerably longer than for that in (B). (D) RNA was extracted from cells and 5 µg total RNA was separated by agarose gel electrophoresis before northern blotting for ß-casein mRNA levels. The blot was reprobed with an 18S cDNA probe. L9 represents mammary tissue extract from day 9 of lactation, used as a control.