α-Br-TMC inhibits both JAK2 and STAT5 phosphorylation.

<p>Ba/F3 cells were pre-treated 30 minutes with the indicated concentrations of TSA and α-Br-TMC (<b>A</b>) or with 10 µM α-Br-TMC and DMSO vehicle (<b>B, C</b>) and further stimulated with 5 ng/mL IL-3 for 30 minutes (<b>A</b>), 15 minutes (<b>B</b>) or 5 minutes (<b>C</b>). Final DMSO concentration was 0.02% in (<b>A</b>) and 0.01% in (<b>B, C</b>). Protein whole cell extracts (<b>panels A, C</b>) or cytosolic (C) and nuclear (N) extracts (<b>panel B</b>) were prepared as described in Materials and Methods and analyzed by Western-blot using antibodies specific for phospho-STAT5 (pSTAT5), phospho-JAK2 (pJAK2), STAT5A, STAT5B, STAT5A and B, JAK2 and α-tubulin (loading control and cytosolic-specific marker). SDS-PAGE in (C) was shorter, explaining why the STAT5 mobility patterns (α-Br-TMC-induced shift and STAT5A and B doublet) are not as apparent as on immunoblots in (A, B).