Western blot using Stat5a (phospho Y694) monoclonal antibody, clone 5F6.F1 (Cat # MAB2097) shows detection of phosphorylated Stat5a (indicated by arrowhead at ~91 KDa) in NK92 cells after 30 min treatment with 1 Ku of IL-2 (lane 2). No reactivity is seen for non-phosphorylated Stat5a in untreated cells (lane 1). The membrane was probed with the primary antibody at a 1 : 1,000 dilution, overnight at 4°C. For detection DyLight™800 conjugated Gt-a-Mouse IgG was used at a 1 : 20,000 dilution for 30 min at room temperature followed by visualization using a VersaDoc™ MP 4000 imaging system (Bio-Rad).
Immunohistochemistry using Stat5a (phospho Y694) monoclonal antibody, clone 5F6.F1 (Cat # MAB2097) shows detection of phosphorylated Stat5a pY694 in human breast tissue (40X). The antibody was used a dilution to 20 ug/mL . The image shows breast epithelium with moderate nuclear staining. Tissue was formalin fixed and paraffin embedded. No pre-treatment of sample was required. The image shows the localization of antibody as the precipitated red signal, with a hematoxylin purple nuclear counterstain. Personal communication, Andrew Elston, Lifespan Biosciences, Seattle, WA.