Journal: Diabetes

Article Title: The Role of TRAPγ/SSR3 in Preproinsulin Translocation Into the Endoplasmic Reticulum.

doi: 10.2337/db21-0638

Figure Lengend Snippet: Figure 1—The TRAPg/SSR3 subunit of the TRAP/SSR complex contributes to the efficiency of recombinant proinsulin biosynthesis. A: Schematic of the TRAP/SSR complex highlights the predominant cytosolic exposure of the TRAPg/SSR3 subunit, including a hypothetical contact with a signal peptide N-terminus during polypeptide translocation (red line). B: Control (Ctrl) (wild-type [WT] 293T cells) and SSR3- KO 293T cells were transfected with untagged human preproinsulin plasmids. At 48 h posttransfection, cell lysates were analyzed by reducing SDS-PAGE gel and immunoblotting with antiproinsulin and anti-SSR1–4 antibodies as indicated. C: Quantitation (mean ± SD) of preproinsulin, proinsulin, and TRAP/SSR subunit protein levels from four independent experiments like that shown in B (normalized to tubulin). *P < 0.05 compared with Ctrl. D: Ctrl and SSR3-KO 293T cells were transfected with human preproinsulin plasmids. At 48 h post- transfection, the cells were pulse labeled with 35S-Met/Cys for the times indicated; this is a representative pulse labeling from three identi- cal experiments. Cell lysates (normalized to trichloroacetic acid–precipitable counts) were subjected to immunoprecipitation with anti- insulin and analyzed by reducing SDS-PAGE and phosphorimaging; both the absolute and relative amounts of recovered preproinsulin and proinsulin are shown in the graph.

Article Snippet: Mouse anti-rat proinsulin (CCI-17) and rabbit polyclonal anti-SSR1 antibodies (NB100-73013 and NBP1-86912, respectively) were from Novus Biologicals (Littleton, CO).

Techniques: Recombinant, Translocation Assay, Control, Transfection, SDS Page, Western Blot, Quantitation Assay, Labeling, Immunoprecipitation

Journal: Frontiers in endocrinology

Article Title: Pharmacological Inhibition of Inositol-Requiring Enzyme 1α RNase Activity Protects Pancreatic Beta Cell and Improves Diabetic Condition in Insulin Mutation-Induced Diabetes.

doi: 10.3389/fendo.2021.749879

Figure Lengend Snippet: FIGURE 5 | STF improves Akita islet b-cell function. (A) Glucose-stimulated insulin secretion of 20 islets isolated from Akita mice treated with STF or vehicle and incubated with 2.5 mM and 16.7 mM glucose. Secreted insulin was measured by ELISA. The data was presented as fold change and normalized with total protein concentration, with the amount of insulin secreted in response to 2.5 mM glucose from vehicle-treated group set to 1.0. (B–E) mRNA levels for indicated genes were analyzed in islets isolated from Akita mice treated with STF or vehicle by qRT-PCR. The results are expressed as fold change and are representative of 3 independent experiments. (F) Proinsulin content measurement by ELISA as detailed in Methods and Materials. (G, H) Proinsulin content and secretion measurement. 20 islets isolated from Akita mice treated with STF or vehicle were incubated with 2.5 mM and 16.7 mM glucose. Secreted proinsulin was measured by ELISA. Proinsulin content measurement by ELISA as detailed in Methods and Materials. The data was presented as fold change and normalized with total protein concentration, with the amount of proinsulin in response to 2.5 mM glucose from vehicle-treated group set to 1.0. (I) Proinsulin misfolding detection by Western blotting under nonreducing condition. Islets were treated with compounds at indicated concentrations for 16 hours. The data is representative of 3 independent experiments. (J–L) Ultrastructure of b-cells in islets isolated from Akita treated with STF as in by transmission electron microscopy. Images at the top panel and the bottom panel were taken at 3,000x and 10,000x, respectively. Arrows point to dark mature insulin granules. *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: The nonreducing gels were incubated in 25 mM dithiothreitol (DTT) solution for 10 min at room temperature before being transferred to PVDFmembranes for 25 min at 4°C at 40 V. Membranes were probed with anti-proinsulin antibody (CCI-17, NOVUS) and HRP-conjugated secondary antibodies (1:3000; Santa Cruz Biotechnology, CA, USA).

Techniques: Cell Function Assay, Isolation, Incubation, Enzyme-linked Immunosorbent Assay, Protein Concentration, Quantitative RT-PCR, Western Blot, Transmission Assay, Electron Microscopy