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

<strong>Supplemental Figure 1.</strong> <strong>Protein levels for proinsulin and controls in lysate and media.</strong> 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. <br> <strong>Supplemental Figure 2. Proinsulin IP analysis by SDS-PAGE and MS/MS</strong> 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). <br> <strong>Supplemental Figure 3. AP-Western validations for selected Proinsulin Interactors identified by AP-MS. </strong>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. <br> <strong>Supplemental Figure 4. Western blot for phospho-eIF2a and total eIF2a. </strong>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. <br> <strong>Supplemental Figure 5. KIF2A versus Insulin expression in single human </strong>b<strong>-cells. </strong>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 &gt;12 and with GCG log2 normalized reads &lt;10. By these criteria, 99 cells were chosen for visualization of plotted INS values overlaid with KIF2A values.