Iblot western detection chromogenic kit

After removing spent media and washing cells with cold phosphate buffered saline (PBS), the cells were incubated with cold Pierce RIPA lysis buffer (Thermo Scientific, Hudson, NH) containing Halt™ Protease Inhibitor Single-Use Cocktail, Halt™ Phosphatase Inhibitor Single-Use Cocktail (Thermo Scientific) and 1 mM dithiothreitol (DTT) for 15 min with occasional swirling. Then, the cells were scraped, homogenized with a 26-gauge needle and vortexed at the highest setting for 1 min; the lysates were cleared by centrifuging at 16,000 g at 4°C for 15 min. Protein concentration was determined with the bicinchoninic acid (BCA) method (BCA Protein Assay - Reducing Agent Compatible; Thermo Scientific). Proteins were separated on NuPAGE 4–12% Bis Tris gel electrophoresis (Life Technologies), and transferred to a nitrocellulose membrane (iBlot Gel Transfer Stacks Nitrocellulose; Life Technologies) using iBlot Gel Transfer Device (Life Technologies). The membrane was probed with monoclonal rabbit antibodies, anti-HMGCR (1:500, ab174830, Abcam Inc., Cambridge, MA), anti-vimentin (1:1000, ab92547, Abcam), and anti-E-cadherin (1:1000, 24E10, Cell Signaling Technology, Beverly, MA). A monoclonal mouse antibody to β-actin (1:500, ab8226, Abcam) was used as a loading control. Immunodetection was performed using the iBlot Western Detection chromogenic kit (Life Technologies).