Mca465ga

Cells were harvested in radioimmunoprecipitation assay buffer (RIPA) containing 1 × protease inhibitor cocktail (cOmplete, 11,697,498,001) and 1% phosphatase inhibitor cocktail II (P5726-5ML, Sigma-Aldrich, St. Louis, MO). Protein separation was achieved under reducing and denaturing conditions using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene difluoride (PVDF) membrane, and immunoblotted with anti-phospho-S726- or anti-phospho-S780-STAT5a antibodies obtained from Abcam (ab128896, ab30649, Cambridge, UK), anti-phospho-Y694-STAT5 antibody (9351S, Cell Signaling Technology, Danvers, MA), anti-STAT5a antibody (sc-1081X, Santa Cruz, Dallas, TX), anti-V5 tag antibody (R96025, Thermo Fisher Scientific), or anti-vinculin antibody (MCA465GA, Bio-Rad, Hercules, CA). HRP conjugated secondary antibodies were obtained from Cell Signaling Technology.

For all IB applications, cells were grown to approximately 80% confluency, washed with ice-cold 1x PBS, and harvested in RIPA (1% NP40, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate pH 7.2, 2 mM EDTA, 50 mM sodium fluoride) supplemented with 5% BME and 1x Laemmli Sample Buffer (161–0747, Bio Rad). For all PRL stimulation experimental lysates, cells were serum-starved for 16–20 h and stimulated at 250 ng/mL PRL (a physiologic level during pregnancy). All blots were derived from the same experiment and processed in tandem. Antibodies used for these studies were obtained from the following sources at the indicated titer: hPRLr ECD (35–9200, Invitrogen, 1:1000), pY-Stat5a (9359S, Cell Signaling, 1:1000), Stat5a (sc-1081, Santa Cruz Biotechnology, 1:1000), pY-Jak2 (3776S, Cell Signaling, 1:500), Jak2 (3230S, Cell Signaling, 1:500), p-p44/42 (9101S, Cell Signaling, 1:1000), p44/42 (9102S, Cell Signaling, 1:1000), pS-Mek (9121S, Cell Signaling, 1:1000), Mek (9122S, Cell Signaling, 1:1000), KRAS (14412S, Cell Signaling, 1:1000), hPRLrI (New England Peptide, 1:5000), pS349-hPRLr (Serge Y. Fuchs, M.D., Ph.D., University of Pennsylvania, 1:100), Vinculin (MCA465GA, Bio-Rad, 1:1000). Densitometry was performed using ImageQuant TL (GE) general image analysis software.