Proinsulin pulse-chase biosynthesis and transport assay

INS-1 832/13 cells were seeded in 12 well plates (500 000 cells/well) and transfected with control siRNAs (against luciferase) or Jagn1 siRNAs. Proinsulin biosynthesis was monitored as described previously [13]. Briefly, the cells were washed once with PBS and incubated in 0.5 ml labelling media consisting of ³⁵[S]-methionine/ cysteine (100 μCi/0.5 ml, Perkin Elmer, NEG022T001MC) in methionine/ cysteine-free DMEM for 20 min at 37 C. The cells were then washed with PBS and incubated on ice for 20 min in lysis buffer (1% Triton X-100, 100 mM KCl, 2 mM EDTA, 20 mM HEPES, pH 7.3, supplemented with protease inhibitors (Roche) and 0.5 mM PMSF. Alternatively, following labelling, the cells were washed with warm PBS and incubated with regular DMEM serum-free media for 30 min at 37 C, then lysed (chase condition). Protein concentration in the lysates was measured and equal amounts of protein (~150 μg)/ condition were incubated with 5 μg of insulin antibody (Santa Cruz) overnight at 4C with rotation. The next day lysates were incubated with Protein A Dynabeads (25 μl) for 3 h at 4 C with rotation. The magnetic beads were washed 3 times with lysis buffer then once with PBS/ 0.1% TX-100. The beads were then incubated with 2X NuPage sample buffer supplemented with 10% β-mercaptoethanol and boiled for 5 min. The sample buffer lysates were resolved on 4–12% Nupage gels, which were subsequently stained with Coomassie blue, washed extensively and dried at 52 C for 3 h. Once dried, the gel was exposed to a Phosphor screen (Amersham Biosciences) and processed using a PhosphorImager (Storm 840, Molecular Dynamics). Three independent experiments were performed and proinsulin band densitometry was quantified using ImageQuant software.