Fig. 2.

(A and B) Transmission electron microscopy with immunogold-labeled IRIS-1 (A), and IRIS-2 (B) antibodies (20-nm particles) and insulin antibodies (10-nm particles) were used to visualize the binding between IRIS-1/2 and insulin granules in INS-1 CD59-knockout (KO) cells overexpressing human IRIS-1 or IRIS-2, after low or high glucose incubation. Colocalization between IRIS proteins and insulin granules in low and high glucose conditions was quantified in C. (D) Isolated healthy human pancreatic islets were divided into two and treated with either low (1 mM), or high (16.7 mM) glucose for 72 h, followed by blotting using specific IRIS-1 antibodies. Quantification of five biological repeats (five different donors) is shown on the Right. (E) IRIS-1 protein levels were assessed by blotting of human pancreatic islets from T2D and healthy donors. Quantification of three technical repeats from nine healthy and 10 T2D donors is shown in F. Lysates of INS-1 cells overexpressing CD59 isoforms, treated with deglycosylating enzymes, indicates lack of glycosylation of IRIS-1 in human pancreatic islets and specificity of the IRIS-1 antibody. (G) IRIS-2 protein levels were assessed by blotting of human pancreatic islets from T2D and healthy donors. Quantification of three technical repeats from nine healhy and 10 T2D donors is shown in H. Statistics (in C): two-way ANOVA with Bonferroni’s posttest; (in D): two-tailed, paired t test; (in F and H): nonparametric Mann–Whitney test. Error bars indicate SD. *P < 0.05, **P < 0.01, and ****P < 0.0001.