Figure A5.

Ba/F3 cells were kept in medium without IL-3 overnight and split in the morning. One half was incubated with 10 ng/mL IL-3 for 40 min (Ba/F3 + IL-3), while the other half was kept in medium without IL-3 (Ba/F3 − IL-3). Thereafter, cells were analyzed for expression of pSTAT5 either by flow cytometry (A) or Western blotting (C). In (A), an Alexa-647-labeled anti-pSTAT5 antibody was used (with IL-3: blue histogram; without IL-3: open blue histogram). mIgG1 Alexa-647 was used as an isotype control (open black histogram) in Ba/F3 + IL-3 cells (A). Human lymphoma cell lines with detectable pSTAT5 levels (KOPT-K1 and MYLA) or no detectable pSTAT5 (HUT78 and HH) were also analyzed for pSTAT5 expression using flow cytometry (B) and Western blotting (D). In Western blot experiments, HSC70 served as a loading control. The columns show the levels of pSTAT5 in percent of HSC70 as analyzed by densitometry. Uncropped blots are shown in Figure S4.