Complete protease inhibitor cocktail

Rat tissue protein in the myocardium of the infarct zone was extracted with lysis buffer (pH 7.5) containing 50 mmol/L Tris‐HCl, 150 mmol/L NaCl, 0.1% sodium dodecyl sulfate, 1% Triton X‐100, 1% Na‐deoxycholate, 1% protease, and complete protease inhibitor cocktail (Sangon Biotech, catalog #C510003). For the whole cell lysate, cells were lysed with a Cell Total Protein Extraction Kit (Sangon Biotech, catalog #C510003) containing a cocktail of phosphatase inhibitors and protease inhibitors. The membrane and cytoplasmic proteins were extracted using a Membrane and Cytoplasmic Protein Extraction Kit (Beyotime, catalog #P0033) according to the manufacturer's instructions.
Primary antibodies for VE‐cadherin, VEGFR‐2, VEGFR‐3, Ras, Erk1, Erk2, Rho, AKT, Dll4, Notch‐1, Notch intracellular domain (NICD), β‐actin, or Na⁺/K⁺‐ATPase were followed by fluorescently labeled anti‐mouse or anti‐rabbit antibodies (Li‐Cor), and the blot was then imaged using the Odyssey infrared imaging system (Li‐Cor). Western blots were quantified using ImageJ software. Protein levels were calculated from the ratio of corresponding protein/β‐actin, and membrane protein levels were calculated from the ratio of corresponding protein/Na⁺/K⁺‐ATPase.