Anti cdk1 antibody

Ten or 50 pmol of precursors or AsOs for miR-31 or their respective controls were transiently transfected into HEPA-1 cells by using Lipofectamine 2000^® (Invitrogen, Carlsbad, CA). After 72 hours, the cells were harvested and homogenized in RIPA buffer (ThermoFisher Scientific, Rockford, IL) to prepare whole cell homogenates. Samples were separated through 10% Tris/glycine/SDS gel electrophoresis and were transferred to a nitrocellulose membrane (ThermoFisher Scientific, Rockford, IL). The membrane was blocked with 5% nonfat milk in blocking buffer and was blotted with 1:3000 anti-CDK1 antibody (Abcam, Cambridge, MA) or 1:5000 anti-GAPDH antibody (Cell Signaling Technology, Danvers, MA). Goat anti-rabbit horseradish peroxidase (GE Healthcare Life Sciences, Pittsburgh, PA) was used as a secondary antibody. The protein bands on the membranes were developed by using a SuperSignal West Femto kit (ThermoFisher Scientific, Rockford, IL). The blots were scanned, and labeling intensities of the bands were quantitated by using ImageJ (National Institutes of Health, USA, http://imagej.nih.gov/ij/). The relative CDK1 protein levels were analyzed through densitometric image analysis and expressed relative to GAPDH levels with the mean ± S.E. of triplicate observations and p < 0.05 based on Student t-test.