Sds page 8 16 tris glycine gel

LCC6 Ctrl and LCC6 IRKD cells and tumor tissue were lysed in ice-cold lysis buffer containing 50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1.25% CHAPS, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 8 mM B-glycerophosphate and Complete Protease Inhibitor Cocktail tablet (Roche, Indianapolis, IN). The protein concentration of the cells and tumors was measured using the BCA protein assay kit (Thermo Scientific, Rockford, IL). 25ug of protein sample was resuspended in 3× loading buffer supplemented with DTT (Cell Signaling Technologies, Danvers, MA). The samples were denatured at 96°C for 5min. The prepared samples were run on SDS-PAGE 8–16% Tris-glycine gel (Invitrogen Life Technologies, Grand Island, NY) and transferred to nitrocellulose membrane. After overnight incubation at 4°C with primary antibodies, the membranes were incubated with secondary antibodies (Li-Cor Biosciences, Lincoln, NE) and scanned using the Li-Cor infrared imaging system. The western bands were quantified using open source Image J software (National Institutes of Health, Bethesda, MD).

MDA-MB-231 cells tumor tissue and Rag/MKR liver tissue were lysed in ice-cold lysis buffer containing 50mM Tris-HCl pH 7.4, 150mM NaCl, 1mM EDTA, 1.25% CHAPS, 1mM sodium orthovanadate, 10mM sodium pyrophosphate, 8mM β-glycerophosphate and Complete Protease Inhibitor Cocktail tablet (Roche, Indianapolis, IN). The protein concentration of the cell and tumor lysates was measured using the BCA protein assay kit (Thermo Scientific, Rockford, IL). Twenty-five micrograms of each protein sample was resuspended in 3× sodium dodecyl sulfate (SDS) loading buffer supplemented with Dithiothreitol (DTT) (Cell Signaling Technologies, Danvers, MA). The samples were denatured at 96°C for 5 min and loaded on an SDS-PAGE 8–16% Tris–glycine gel (Invitrogen Life Technologies, Carlsbad, CA) and transferred onto nitrocellulose membrane. After overnight incubation at 4°C with primary antibodies, the membrane was incubated with secondary antibodies (Li-Cor Biosciences, Lincoln, NE) and scanned using the Li-Cor infrared imaging system. The protein bands were quantified using Image Studio Lite software (Li-Cor Biosciences, Lincoln, NE).