4.6. Western Blot Analysis of Expression of pSTAT5 in Ba/F3 Cells

To confirm the selective effects of JAK2 or STAT5 targeting drugs, Western blot experiments were performed using the pSTAT5 antibody Tyr694 on Ba/F3 and Ba/F3-MPL cells engineered to express JAK2 V617F and CALRdel52, respectively. Ba/F3 cells were generated as described [71,72]. Cells were incubated with drugs targeting JAK2 (AZD1480, TG101348, ruxolitinib, R763; 1–5 µM) or STAT5 (piceatannol, pimozide; 10–45 µM) for 4 h. Then, pSTAT5 expression was analyzed by Western blotting essentially as described [49]. Nitrocellulose membranes (0.45 µm Amersham Protran; order number: 10600002; GE Healthcare, Buckinghamshire, UK) were incubated with the following antibodies at the dilution indicated: polyclonal rabbit anti-phospho-STAT5 (Y694) antibody (order number: 71-6900; 1:1000; Invitrogen, Camarillo, CA, USA), monoclonal mouse anti-STAT5 antibody 89/Stat5 (order number: 610191; 1:1000; BD Biosciences), mouse anti-HSC70 monoclonal antibody B-6 (order number: SC-7298; 1:10,000; Santa Cruz, St. Louis, MO, USA), IRDye^® 680RD goat anti-rabbit IgG (order number: 925-68071; 1:10000; LI-COR, Lincoln, NE, USA), and IRDye^® 800CW goat anti-mouse IgG (order number: 925-32210; 1:10000; LI-COR). pSTAT5 levels were quantified by densitometry and expressed as the pSTAT5/loading control ratio normalized to control (untreated) cells.