Figure 1. Glucose stimulates the recruitment of mRNAs encoding secretory membrane proteins to the ER: subcellular fractionation by consecutive centrifugation.

MIN6 cells were preincubated in KRB containing 0.5 or 2 mM glucose for 1 h followed by incubation for a further period of 1 h at the same glucose concentration or 20 mM glucose. Cells were fractionated by consecutive centrifugation resulting in a membrane fraction (P6), a residual membrane fraction (P18), a polysome fraction (P200) and a supernatant fraction containing free mRNAs (S200); 10% refers to 10% of total RNA/protein. (ai, bi) RNA isolated from each fraction was run on a 1% agarose formaldehyde gel, transferred on to a nylon membrane and probed for proinsulin (PI), PC2, CPH and actin mRNAs. (aii, bii) The percentage increase in mRNA levels at the ER in response to high glucose was determined by quantification of bands from Northern-blot analysis using ImageJ. The error bars indicate the S.E.M. (n=7). Significant differences are indicated by *P<0.05 (two sample t test). (c) Protein samples from each fraction (8% of the P6/P18/P200 fraction and 1% of the S200 fraction) were run on SDS/PAGE (12.5% polyacrylamide) and subjected to immunoblotting with anti-ERK2, calreticulin and SRP54 antibodies. Detection was by enhanced chemiluminescence. (d) Time course of the recruitment of proinsulin mRNA to the ER. MIN6 cells were incubated in KRB containing 0.5 mM glucose followed by incubation for the times indicated in KRB containing 20 mM glucose. Results presented are representative of three separate experiments.