Figure 2. Glucose stimulates the recruitment of mRNAs encoding secretory membrane proteins to the ER: subcellular fractionation using the digitonin method.

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 permeabilized with digitonin and pelleted resulting in a membrane fraction (Mem) and a cytosolic fraction (Cyt). The 10% refers to 10% of total RNA/protein. RNA was isolated from each fraction and run on a 1% agarose formaldehyde gel, transferred on to a nylon membrane and probed for proinsulin (PI), PC2, CPH and actin mRNAs. The intensities of the bands were quantified using ImageJ and the mRNA levels were expressed as a percentage of total mRNA. (b) Protein samples from each fraction were run on SDS/PAGE (20% of membrane fraction and 20% of cytosolic fraction) and subjected to immunoblotting with anti-ERK2, calreticulin and SRP54 antibodies. Detection was by enhanced chemiluminescence. The results presented are representative of three separate experiments.