Materials and methods

Cell lines and transient transfections
The human breast cancer cell lines SKBr3, BT-20, BT-549, MDA-MB-468, and T47D were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were maintained in DMEM plus 10% FCS and were passaged twice per week. Mouse embryo fibroblasts (MEF5-/-) from STAT5a/b knockout mice (provided by Dr J Ihle, St Jude Children's Hospital, Memphis, TN, USA) were passaged twice per week and maintained in DMEM plus 10% FCS. Cells were transfected with STAT constructs [23], Brk constructs (generous gift from Dr C Lange, University of Minnesota, Minneapolis, MN, USA), and c-Src constructs as previously described [24], using LipofectAMINE and PLUS reagent according to the manufacturer's instructions (Invitrogen, Gaithersburg, MD, USA).

Reagents
The polyclonal STAT5a-specific and STAT5b-specific antibodies were developed in our laboratory, as previously described [23]. Polyclonal anti-STAT3 antibodies, polyclonal anti-Brk antibodies, and monoclonal antiphosphotyrosine antibodies (PY-99) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The monoclonal anti-β-actin antibody was from Sigma (St Louis, MO, USA). The antiphospho-STAT5a/b (Y694/Y699) antibody was developed in conjunction with Aves Labs (Tigand, OR, USA) as described elsewhere (EM Fox, T Bernaciak, J Wen, A Weaver, M Shupnik, CM Silva - unpublished data). The protease inhibitor cocktail was from Calbiochem (San Diego, CA, USA). The acrylamide was obtained from Bio-Rad (Hercules, CA, USA), and the prestained molecular weight marker was from Invitrogen. Except as noted, other reagents were of either reagent grade or molecular biological grade from Sigma.

Immunoprecipitations and immunoblotting
Cells were lysed in 150 mM NaCl, 5 mM ethylenediamine tetraacetic acid, 1% Triton X-100, 1% deoxycholate, 50 mM Tris (pH 7.4), containing protease inhibitors and phosphatase inhibitors. Lysates were incubated with the indicated antibody overnight at 4°C, and protein A-agarose (Santa Cruz Biotechnology) was added for an additional 1 hour at 4°C. Agarose pellets were washed three times in detergent lysis buffer, and the bound proteins were removed by boiling in 1 × Laemmli buffer. Proteins were separated on polyacrylamide gels and were analyzed as previously described [12].

Luciferase assay
MEF5-/- and BT-549 cells were transfected with the Spi2.1-containing luciferase reporter plasmid. Forty-eight hours post transfection, lysates were prepared and luciferase activity was measured [24]. The luciferase values (arbitrary units), as measured by a Berthold Luminometer (Berthold, Oak Ridge, TN, USA), were normalized to total protein.

Recombinant protein
STAT5b or Y699F cDNA was cloned into the pGEX4T-1 plasmid (Amersham, Piscataway, NJ, USA) using the EcoRI and NotI restriction sites. The GST, GST-STAT5b, and GST-Y699F plasmids were generated and transformed into Escherichia coli BL21. Protein expression was induced by 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG in Luria broth (LB) broth at 18°C for 18 hours. Bacterial cells were lysed in 1 × PBS containing 1 mM ethylenediamine tetraacetic acid, 5 mM dithiothreitol, 1.5% sarkosyl, 1% Triton, 2 mM phenylmethylsulfonyl fluoride (PMSF), and 1 μg/ml pepstatin followed by sonication. GST, GST-wtSTAT5b, and GST-Y699F were isolated using glutathione agarose beads (Sigma) following the manufacturer's instructions. Following elution with excess glutathione (50 mM Tris–HCl/10 mM glutathione, pH 8.0), the recombinant proteins were dialyzed in 1 × Tris-buffered saline/10% glycerol. Protein concentration was determined by the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA).

In vitro kinase assay
Purified recombinant Brk (Upstate, Billerica, MA, USA) and purified recombinant GST, GST-STAT5b, or GST-Y699F were incubated with 20 nM ATP in reaction buffer (100 mM Tris–HCl, pH 7.4, 125 mM MgCl2, 25 mM MnCl2, 2 mM ethylene glycol tetraacetic acid (EGTA), 0.25 mM NaVO4, 2 mM dithiothreitol) at 30°C for 30 minutes. An equal volume of 2 × Laemmli was added to end the reaction. Phosphorylation of STAT5b was analyzed by immunoblotting with our specific phospho-Y694/Y699 STAT5a/b antibody.

RNA isolation and reverse transcriptase polymerase chain reaction
Total RNA was isolated from the breast cancer cell lines using the RNeasy mini kit (Qiagen, Valencia, CA, USA) following the manufacturer's instructions. The cDNA was generated using iScript cDNA synthesis (Bio-Rad). The cDNA was amplified using primers for Brk or β-actin as described by Kasprzycka and colleagues [25].

Small interfering RNA methodology
Knockdown of Brk and/or STAT5b was performed using the siGenome SMARTpool duplex (Dharmacon, Lafayette, CO, USA) transfected with Oligofectamine (Invitrogen) according to the manufacturer's instructions.

DNA synthesis assay
Following 48 hours of transfection with siRNA, SKBr3 cells or BT-20 cells were serum starved for an additional 18 hours and were then incubated with 100 μM bromodeoxyuridine (BrdU) for 6 hours. Cells were fixed and permeabilized as previously described [12]. Cells were blocked in 20% goat serum/PBS for 20 minutes at 37°C, and then were incubated with anti-BrdU-Alexa-Fluor 594 (Molecular Probes, Carlsbad, CA, USA) for 1 hour at 37°C. BrdU incorporation was visualized using a Leica DM RBE Fluorescence microscope (model RS232C; Leica Microsystems, Bannockburn, IL, USA).