PRL has the potential to directly signal to LKB1 in MDA-MB-231 cells. (A) The PRLR LF is expressed at the mRNA level in representative breast cancer cells including MDA-MB-231 cells and 184B5 normal breast epithelial cells, while levels are close to undetectable in A549 lung cancer cells, as assessed by quantitative real time PCR. (B) Various isoforms of the PRLR are potentially expressed at the protein level in 184B5, MCF-7, and MDA-MB-231 cells. The LF migrates at the expected molecular weight of 85-90 kDa, similar to the band obtained in T47D cells, which express high levels of the LF, and (C) is comparable to migration in CHO-K1 cells transiently transfected with an expression vector encoding the LF of PRLR. (D) Representative Western blots of a time-course demonstrating that JAK2, STAT3, and STAT5 are phosphorylated in MDA-MB-231 cells cultured with 100 ng/mL of PRL for 24 hr. (E) Co-immunoprecipitations (IPs) were carried out using equal amounts of total cell lysates followed by Western blotting (WB). IPs with total JAK2 followed by WB with phospho- and total JAK2 were performed on lysates from 184B5, MCF-7, and MDA-MB-231 cells. I: 10% of total non-IP lysate or “input” as a positive control, -: no treatment, +: treated with 100 ng/mL of PRL for 24 hr, ++: pre-treated with 5 μM WP1066 for 2 hr followed by the addition of PRL for 24 hr. (F) PRL also temporally induced inactivation (phosphorylation) of ACC.