Panel A: Coimmunoprecipitation studies: Herein, INS 832/13 cells were incubated in the presence of low glucose [LG, 2.5 mM] or high glucose [HG, 20 mM] for 30 min at 37°C. ARNO was immunoprecipitated in the lysates using a specific antibody as described in Methods. The immunoprecipitates were separated by SDS-PAGE and probed for Arf6. A representative blot from three studies is shown.
Panel B: Data from multiple studies shown in Panel A are analyzed densitometrically and expressed as densitometric units and are mean ± SEM. * represents p <0.05 vs. low glucose.
Panel C: Immunofluorescence studies using confocal microscopy: INS 832/13 cells were cultured on coverslips and cultured overnight prior to the incubation with either 2.5 mM or 20 mM glucose for 30 min at 37°C. The cells were fixed in 4% paraformaldehyde solution in PBS for 15 min and permeabilized using 0.2% triton X-100 for 15 min. Fixed cells were examined for Arf6 [stained in green] and ARNO [stained in red] as described under Methods.