Anti stat5 antibody

ChIP assays were performed using the EZ-ChIP kit (Millipore) according to the manufacturer's instructions with minor modifications. Anti-FLAG M2 affinity gel (Sigma, A2220) and anti-STAT5 antibody (Santa Cruz, sc-835) were used overnight at 4°C to immunoprecipitate flag-CUZD1 and STAT5, respectively. Normal mouse IgG (Santa Cruz, sc-2027) immunoprecipitation served as a negative control. Protein/DNA complexes were eluted, crosslinks were reversed and purified DNA was analyzed for enrichment in sequences of interest using qPCR.

Formalin-fixed paraffin-embedded mouse spleens were cut into 10 μm sections. The tissue slides were deparaffinized, rehydrated in decreasing alcohol concentrations and incubated in 3% H₂O₂ for 10 min to block endogenous peroxidase activity. Antigen retrieval was performed at 98°C for 50 min in 10 mM HIER citrate buffer pH 6 (Zytomed Systems, Berlin, Germany) in the water bath. Nonspecific protein binding was blocked with 2% goat serum and 2% BSA in PBS for 30 min. The slides were then incubated with anti-STAT5 antibody (1:200, Santa Cruz Biotechnology) overnight at 4°C. After incubation for one hour with the second anti-rabbit antibody (EnVision+ System-HRP labelled Polymer, DAKO, Hamburg, Germany) the signal was visualized by incubating the slides with AEC substrate (DAKO) and the nuclei were counterstained with hematoxylin (Carl Roth, Karlsruhe, Germany). Spleen sections from mice with FLT3-ITD-driven leukemia or transplanted with mock-transduced HSPCs were used as positive and negative controls, respectively. Staining with the secondary antibody served as further negative control

Protein lysates were prepared from tissues crushed to powder under liquid nitrogen (~ 20 mg) using 300 μL of tissue lysis buffer (50 mm Tris 7.3, 150 mm NaCl, 1 mm EDTA pH 8.1, 1.5 mm MgCl₂, 10% glycerol, 1% Triton X‐100, 10 mm Na₄P₂O₇, 100 mm NaF, 1 mm Na₃VO₄, 1 mm phenylmethanesulfonyl fluoride, 5 μg·mL⁻¹ aprotinin, and 5 μg·mL⁻¹ leupeptin). Lysates were resolved under reducing conditions by SDS/PAGE and transferred to nitrocellulose membranes (Amersham Biosciences), followed by blocking with 2% BSA. Membranes were immunoblotted (Table 1) with anti‐phospho‐STAT5 antibody (Y694; Cell Signaling; 9351L) (1 : 1000), which reacts with both phosphorylated Y694 in STAT5A and Y699 in STAT5B; anti‐STAT5 antibody (Santa Cruz Biotechnology; sc‐835) (1 : 1000); anti‐GHR (polyclonal anti‐GHR_cytAL‐47; against the intracellular domain of GHR) 26 (1 : 1000); anti‐PRLR (anti‐PRLR_cytAL‐84; against the human PRLR ICD) 27 (1 : 1000); anti‐PRL‐R (H‐300) (Santa Cruz Biotechnology; sc‐20992); and anti‐JAK2 (anti‐JAK2_AL‐33) 28 (1 : 1000). Densitometry was performed using UVP Software 8.0.