Determination of downstream leptin signaling pathway sequence. The Sca-1+/CD45-/CD34+ cells were seeded as 1 × 107/10ml/dish. Cells were then pre-incubated with 200 μM of the PI3 Kinase inhibitor LY294002, 10 μM of the Akt inhibitor IV, and 10 μM of the ERK inhibitor U-0126 for 1 h. After 1 hr, cell culture media was removed and fresh media containing leptin (1000 ng/ml) and the same inhibitors were added for additional 30 min. Total cellular proteins were extracted and subjected to western blot analysis with (A). anti-phospho-Akt (B). anti-phospho-p44/42 ERK, and (C), anti-phospho-STAT3 (Ser727) and anti-phospho-STAT3 (Tyr705) antibody as indicated. Bands were detected using the ECL system. Afterward, blots were stripped and reprobed with anti-STAT3, anti-PI3K p85, anti-Akt (pan), and anti-p44/42 ERK antibody for assessment of protein loading. Results shown are representative of three independent experiments.