Figure 5.

Co-immunoprecipitation and quantification of ZnT8. A, ZnT8 probed by mAb20 immunoblotting using liposomes from 1 × 10⁶ cells before and after they were bound to mAb20- or IgG-beads followed by SDS elution. B, co-immunoprecipitation of ZnT8 and organelle markers. ZnT8-liposomes were captured by mAb20 beads, eluted, and then probed by an antibody to BAP31, IA2, TMED3, VAMP2, or Golgin97 as indicated. Prebound liposomes and IgG-bead bound liposomes were used as a positive and negative control, respectively. Of note, gel loading of prebound liposomes was half of the mAb20-bead–bound liposomes. C, quantification of ER-resident ZnT8. Top, representative mAb20 immunoblotting of liposomes before and after incubation with anti-BAP31-beads. Liposomes in the flow-through and SDS-elution of BAP31-beads are marked as unbound and bound, respectively. Bottom, densitometric quantification of ZnT8 intensity of total liposomes and unbound and bound fractions. ZnT8 intensities were normalized to the intensity of total liposomes on the same immunoblot. Error bars, S.E. of four independent experiments. D, cytokine-induced reduction of ER-resident ZnT8. Top, a representative mAb20 immunoblotting of liposomes from an equal number of EndoC-βH1 cells (1 × 10⁶). Ctr and Cyt, liposomes from untreated control and cytokine-treated cells, respectively. Ctr-bound and Cyt-bound are captured Ctr- and Cyt-liposomes on anti-BAP31-beads with SDS elution. Bottom, densitometric quantification of ZnT8 intensities with normalization to the Ctr intensity on the same immunoblot. Error bars, S.E. from four independent experiments. E, confocal microscopy imaging of EndoC-βH1 cells with and without exposure to 1× cytokine mixture for 24 h, which were then co-immunostained with mouse mAb20 and a rabbit anti-insulin pAb. Scale bars, 5 μm.