Phospho stat5

Proteins from MPN-derived MNC were isolated and processed as previously reported [9 (link)]. Equal amounts of protein (30 μg) were run on polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 4% milk (Serva Electrophoresis GmbH, Heidelberg, Germany) for 1 h at room temperature and probed with primary antibodies directed against HIF-1α (Elabscience, Wuhan, China), VEGF (Elabscience), eNOS (Elabscience), β-actin (R&D Systems, Inc, Minneapolis, Minnesota), phospho-STAT5 (R&D Systems), STAT5 (R&D Systems), phospho-AKT (R&D Systems), AKT (R&D Systems), pmTOR (Cell Signaling Technology Inc., Beverly, USA) and mTOR (Cell Signalling Technology). Peroxidase-conjugated goat anti-rabbit immunoglobulin (R&D Systems) was used as a secondary antibody, except goat anti-mouse immunoglobulin (R&D Systems) was used for β-actin. The protein levels were imaged with a ChemiDoc Imaging System (Bio-Rad Laboratories, Hercules, CA, USA) and estimated by densitometric scanning of the blots using the Image Lab (Bio-Rad Laboratories, Inc. Version 6.0.0.25) software tool and normalized to β-actin.

Protein isolation and Western blotting were performed as described earlier.20 Briefly, 28 μg of protein concentrate was mixed with 4× Laemmli buffer (8% SDS, 40% glycerol, 20% 2‐mercaptoethanol, 0.008% bromphenol blue, 0.25 mol/L Tris‐HCl, all from Sigma‐Aldrich) and incubated for 3 minutes at 95°C. Samples were separated by SDS‐PAGE and transferred to a PVDF membrane (Amersham Pharmacia Biotech). After 2 hours of blocking with TBST buffer containing 3% of bovine serum albumin (Carl Roth) at room temperature, the membranes were incubated overnight at 4°C with specific primary antibodies for Erk1/2 (1:10 000; Cell Signaling Technology), phospho‐Erk1/2 (1:8000; Cell Signaling Technology), Akt (1:2000; Cell Signaling Technology), phospho‐Akt (1:1000; Cell Signaling Technology), STAT5 (1:1000; R&D Systems) and phospho‐STAT5 (1:1000; R&D Systems). Signals from peroxidase‐conjugated secondary antibodies (anti‐rabbit; 1:20 000; Cell Signaling Technology) were detected using the ECL kit (ECL‐Plus Western blotting Detection Reagents; Amersham Pharmacia Biotech). Membranes were exposed to X‐ray‐films and intensities of the respective protein bands were analysed using the imagej software 1.41 (National Institutes of Health NIH).