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Western blot analysis of GAPDH was performed by loading 20 µg of A549 (lane1), MCF7 (lane2), U-87 MG (lane3) and HeLa (lane4) cell lysate using Novex® NuPAGE® 4-12 % Bis-Tris gel (NP0321BOX), XCell SureLock™ Electrophoresis System (EI0002), Novex® Sharp Pre-Stained Protein Standard (LC5800), and iBlot® Dry Blotting System (IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5 % skim milk for 1 hour at room temperature. GAPDH was detected at ~37 kDa using GAPDH Mouse Monoclonal Antibody (A21994) at 0.1-0.2 µg/mL in 2.5 % skim milk at 4°C overnight on a rocking platform. Goat Anti-Mouse IgG - HRP Secondary Antibody (626520) at 1:4000 dilution was used and chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (WP20005).
