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Western blot analysis was performed on whole cell extracts (30 µg lysate) of A-549 (Lane 1), A-431 (Lane 2), T-47D (Lane 3) and MCF-7 (Lane4). The blots were probed with Anti-VEGFR Rabbit Polyclonal Antibody (Product## 720043, 0.5-1ug/ml) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal? Secondary Antibody, HRP conjugate (Product ## A27036, 0.4 ug/ml, 1:2500 dilution). Two isoforms showing 150 and 51kDa band corresponding to VEGFR was observed in cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris gel (Product ## NP0321BOX), XCell SureLock? Electrophoresis System (Product ## EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product ## LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product ## IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product ## WP20005).
