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. 2005 Oct 10;391(Pt 2):291–300. doi: 10.1042/BJ20050468

Figure 5. Glucose stimulates the recruitment of ribosomes on to membrane-bound proinsulin mRNA.

Figure 5

MIN6 cells were preincubated in KRB containing 2 mM glucose for 1 h followed by incubation in KRB containing 2 or 20 mM glucose for a further period of 1 h. (a) Cells were then lysed and fractionated by consecutive centrifugation. The membrane fractions were layered on to 20–50% sucrose gradients, centrifuged and fractionated from left (fraction 1) to right (fraction 10). RNA was isolated from each fraction and run on a 1% agarose formaldehyde gel. RNA was then transferred on to a nylon membrane and probed for proinsulin (PI), PC2 and CPH mRNAs. (b) Cells were then lysed and fractionated by the digitonin cell permeabilization method (using modified polysome buffer containing 130 mM KCl), resulting in a membrane fraction (Mem) and a cytosolic fraction (Cyt). The membrane fraction was consecutively washed with polysome buffer containing 250 mM KCl (250 mM wash) and 500 mM KCl (500 mM wash). RNA isolated from each fraction and washed was run on a 1% agarose formaldehyde gel, transferred on to a nylon membrane and probed for proinsulin (PI) mRNA; 10% refers to 10% of total RNA.