2.8. Western blot assay

After transfection, the lung cancer cells were cultured until reached the logarithmic growth phase, and then the total protein was extracted as suggested by the protein extraction steps. The total protein concentration was measured by 2,2′-bicinchoninic acid (BCA) and the protein was transferred onto nitrocellulose membrane after subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Following mounting in 5% skimmed milk powder at room temperature for 1.5 hours, the membrane was washed with Tris-buffered saline Tween-20 (TBST), added with anti-DICER1 (1:1000) and β-actin (1:500) antibodies and incubated at 4°C overnight. After washing with TBST, horseradish peroxidase-labeled goat antirabbit secondary antibody (1:5000) was added into membrane for incubation at 37°C for 1 hour. After that, the membrane was washed with TBST again, and then developed via chemiluminescence. Images were acquired by ImageJ software with β-actin band as an internal reference and changes of DICER1 protein expression were observed. The experiment was repeated 3 times.