This is more difficult to occur in the RNA extracted from tumor cells

7500 rpm for 5 min at 4C. RNA was dissolve in RNAse free water. Determination of glucose uptake by 20142041 the mice GLS The rate of glucose uptake by the GLS was determined by using non metabolizable 2-deoxy-D-14C glucose as described. Immunohistochemical localization of Glut-4 in the mice hepatocytes The mice liver “chunks” were sliced into 6-8 nm sections in a cryostat to demonstrate the presence of Glut-4 in the hepatocytes. These sections were incubated with Glut-4 antibody and identified by using fluorescent tagged anti-rabbit immunoglobulin G-alkaline phosphatase as described. Following the immunohistochemistry, sections were imaged by using a fluorescent microscope attached to a high resolution digital colour camera which photographically recorded the non-weighted images of the sections. Separation of plant ribosomes Bael leaf around 10 gm was taken and washed to remove debris twice and rinsed in distilled water and homogenized and later centrifuged at 5000 rpm to remove the debris, the supernatant was taken, and centrifuged at 13,000 x g. The supernatant was layered on top of a 1mM sucrose cushion and centrifuged at 200,000 x g to pellet ribosomes as described. Identification of Glut-4 by immunoblot and its quantitation by enzyme linked immunosorbent assay in the supernatant of the glucose treated ABT-578 disrupted GLS The amounts of Glut-4 synthesized in the crude supernatants of GLS incubated with different concentrations of glucose were identified by immunoblot and quantitated by ELISA by using Glut-4 antibody. The Enzyme linked immunosorbent assay was performed as described. Briefly, Glut-4 was incubated with an equal volume of phosphate buffer saline in an assay plate overnight in 4C. Nonspecific binding was blocked by 5% bovine serum albumin in the 15601771 same buffer. The samples were then washed with PBS containing Tween-20, and incubated for 2h with diluted primary antibody in PBS obtained from Santa Cruz biotech. The samples were next washed with PBS-T20 and incubated with diluted goat antirabbit IgG-alkaline phosphatase in the same buffer for 1h. After washing they were incubated with p-nitrophenyl phosphate in carbonate buffer containing 10mM MgCl2. The development of colour was determined at 405nm. The amount In vito translation of Glut-4 mRNA RNA was isolated by Trizol-chloroform method from GLS described above. Briefly the mRNAs were incubated with ribosomal preparation, mixture of all 20 amino acids and 2mM ATP as described. After 6 h the reaction mixture was centrifuged at 10,000 g at 0C for 10 min. The supernatant was used for the determination of Glut-4 by ELISA as described above. Immunoblot analysis of Glut-4 in the GLS of mice hepatocytes Typically, the pelleted fraction from the disrupted GLS as described above was collected. The presence of Glut-4 in the pelleted fraction of GLS of mice hepatocytes treated with different amounts of glucose and incubated for 30 min at 37C. The presence of Glut-4 in the fraction was identified by immunoblot technique. The samples were subjected to SDSpolyacrylamide gel electrophoresis and stained with 3 Glucose Transporter-4 in Mice Hepatocytes doi: 10.1371/journal.pone.0081935.g001 Coomassie brilliant blue. An identical SDS gel that was not stained with Coomassie brilliant blue also prepared. The protein bands were next transfer to a nitrocellulose membrane, and Glut-4 was subsequently identified in the nitrocellulose membrane by using fluorescent tagged Glut-4 antibody. Determination of the gluc