Human l493 array

Three independent controls and miR-7-treated tumor samples were used for the protein arrays, according to the standard operating procedures of the Raybio (USA) human L493 array (catalog no. AAH-BLG-2-2). Total protein was extracted from 250 mg of tissues with ice-cold cell & tissue protein extraction reagent (KangChen Bio-tech, China, catalog no. KC-415), which contained the inhibitors for protein degradation (5 μL of protease inhibitor cocktail, 5 μL of PMSF, and 5 μL of phosphotase cocktail were added into 1 mL of protein extraction reagent). The protein concentration was determined by a BCA protein assay kit (KangChen Bio-tech, catalog no. KC-430). Protein array membranes were blocked in blocking buffer for 30 min and then incubated with samples at room temperature for 1–2 h, then decanted, and membranes were washed with washing buffer. After that, membranes were incubated with diluted biotin-conjugated antibodies at room temperature for 1–2 h. The membranes were washed with washing buffer and reacted with streptavidin-conjugated fluor at room temperature. Membranes were then washed thoroughly and scanned by an Axon scanner. The intensities of signals were quantified by densitometry. Raw intensities were revised by background and normalized by median. A 2-fold change in protein expression was calculated.25 (link), 26 , 27 (link)