Supplementary Figures and Tables - Inflammatory Cytokines Rewire the Proinsulin Interaction in Human Islets - Tran et. al. 2022

Supplemental Figure 1. Protein levels for proinsulin and controls in lysate and media. A) Western blot analysis of expression in lysate and media; Vinculin, Indoleamine 2,3-Dioxygenase 1 (IDO1) and Guanylate Binding Protein 5 (GBP5) and proinsulin. Inset is second Western blot for proinsulin in media samples B) Intracellular proinsulin measured by ELISA and normalized to lysate GAPDH. Supplemental Figure 2. Proinsulin IP analysis by SDS-PAGE and MS/MS A) Western blot and silver stain of proinsulin immunoprecipitations from human islets +/- cytokines. B) Total MS/MS counts from proinsulin AP/MS (blue) and IgG AP-MS of six human islets +/- cytokines. C) The total number of proteins identified by AP-MS in distinct islet samples labeled Exp. 6,7,10,13,14,15. For each islet sample there are four IP conditions: Untreated/proinsulin IP, Untreated/IgG IP, Cytokine treated/proinsulin IP, and Cytokine treated/IgG IP. Note that biological samples 13-15 each have two technical replicate MS runs each (R1 and R2). Supplemental Figure 3. AP-Western validations for selected Proinsulin Interactors identified by AP-MS. Human islet lysates were immunoprecipitated with antibodies to proinsulin (or IgG), followed by Western blot for ERGIC1, Ataxin-2, ERDJ3, QSOX1, Calnexin, BiP and proinsulin. Men=Menadione, Cyto=Cytokines, M=Markers. Supplemental Figure 4. Western blot for phospho-eIF2a and total eIF2a. Human islets, untreated (CON) or cytokine treated (CYTO) were immunoblotted for eIF2a phosphorylated at Ser51 (peIF2 a) and for total eIF2a. MIN6 cells treated with Thapsigargin (Tg) were used as a positive control for peIF2a. GAPDH served as a loading control. Supplemental Figure 5. KIF2A versus Insulin expression in single human b-cells. For single-cell RNA-Seq data obtained from Gene Expression Omnibus Repository (accession number GSE83139) the normalized reads for insulin (INS), glucagon (GCG), and KIF2A were log2 transformed. We defined b-cell identity as cells with INS log2 normalized reads >12 and with GCG log2 normalized reads