Leptin–induced MCP-1 expression is inhibited by JAK-2 inhibitor, PI3K inhibitor, ERK inhibitor and Akt inhibitor. The Sca-1+/CD45-/CD34+ cells were seeded as 1 ×107/10ml/dish in a 100 mm × 20 mm culture dish. The cells were pre-incubated with 50 μM of the JAK 2 inhibitor AG490 for 1 h. After 1 hr, cell culture media was removed and fresh media containing leptin (1000 ng/ml) and 50 μM of the JAK2 inhibitor was added for additional 30 minutes. Total cellular proteins were extracted and subjected to western blot analysis with (A). anti-phospho-PI3K, (B). anti-phospho-Akt (C). anti-phospho-p44/42 ERK, and (D), 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.