**2. Problem statement**

Studies from our laboratory [1] and from others [2] have demonstrated that three specific isoforms of the PRLR are expressed in both normal and cancerous breast cells and tissues. We recently developed and characterized PRLR isoform specific polyclonal antibodies that

© 2013 Ginsburg et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

revealed that the three isoforms, LF, SF1a, and SF1b, are differentially expressed in ductal and lobular carcinomas [5]. These two most common histological types of breast cancer originate from the terminal ductal lobular unit and may be difficult to classify. However, distinct differences were observed in PRLR expression in normal, benign, and malignant breast tissue which may have prognostic significance [10, 11]. The development and characterization of PRLR isoform specific monoclonal antibodies will provide a near limitless supply of reagent to continue to examine how and where these isoforms interact both in normal breast development and in breast cancer.

Prolactin Receptor Isoforms in Human Breast Cancer 197

Antibodies (Burlington, VT) for generation of mouse monoclonal antibodies. Initially, 120 clones were screened by Western blot as described above using CHO-K1 cells expressing LF with the exception that the antibodies were diluted 1:100 in 5% non-fat dairy milk (diluted in TBST+0.1% sodium azide) and incubated overnight at 4o C. Secondary antibody and imaging were essentially as described above. Supernatants of positively reacting clones were re-screened by Western blot and examined by fluorescence microscopy (to confirm appropriate localization) prior to selection for production. In total, 10 positive clones were selected for this process, 8 of which produced IgG. Antisera was purified as described for SF1a and SF1b antibodies. Final pools of antibody were produced and protein concentration

Chinese hamster ovary cells (CHO-K1, ATCC, Manassas, VA) were maintained in -MEM (Invitrogen, Gaithersburg, MD) supplemented with 5% fetal bovine serum (FBS, Invitrogen) and penicillin/streptomycin (100 U/ml and 100 g/ml respectively, Invitrogen). Transfections were performed using FuGENE 6 (Roche Applied Science, Indianapolis, IN) at a ratio of 1 g DNA to 3 l FuGENE. The PRLR isoform specific cDNA constructs were previously described [1]. Cells were transfected for 48 hr, then allowed to grow for an additional 48 hr. T47D, ZR75-1, MDA-MB-231, MDA-MB-468, MCF7, and SK-BR-3 breast cancer cell lines were obtained from ATCC. T47D, ZR75-1, MDA-MB231, and MDA-MB468 cells were maintained in RPMI1640 (Invitrogen). MCF7 cells were grown in DMEM and SK-BR-3 cells were maintained in McCoys 5a media. All media were supplemented with 5% heat inactivated FBS, 10 g/ml bovine insulin (Sigma, St. Louis, MO), and penicillin-streptomycin (100 U/ml and 100 g/ml respectively, Invitrogen). Where indicated, breast cancer cell lines were plated on 8-well glass chamber slides (Nunc, Rochester, NY) in normal growth media, and allowed to attach overnight. The media were replaced and the cells were treated for the indicated times with 500 ng/ml recombinant human PRL in media where the FBS was replaced with 1% charcoal stripped serum, then fixed in 10% normal buffered formalin prior to Duolink

All cells were maintained at 37 °C in a humidified atmosphere with 5% CO2. Cells were passaged using trypsinization (0.05% trypsin-EDTA, Invitrogen) and counted on a

Transfected CHO cells were collected and whole cell lysates were prepared in Complete Buffer (Roche Applied Science) according to the manufacturer's instructions. Total protein was estimated according to Bradford [12]. Protein (100 g) was subjected to 10-20% SDS-PAGE (Invitrogen). Proteins were transferred to nitrocellulose membrane and probed with PRLR isoform specific antibodies (10 g/ml for LF, 6 g/ml for SF1a or SF1b). Reactivity was detected using ECL Plus (GE Healthcare Life Science, Pittsburgh, PA). Molecular size

determinations were made using BenchMark Protein Ladder (Invitrogen).

determined by the Pierce 660nm assay.

**4.2. Cell culture and transfection** 

hemocytometer using trypan blue exclusion

**4.3. Western blot analysis** 

assay.
