Journal: Cell reports

Article Title: Amino acids-Rab1A-mTORC1 signaling controls whole-body glucose homeostasis

doi: 10.1016/j.celrep.2021.108830

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-Proinsulin (253627) , Novus biologicals , Cat# MAB13361; RRID: AB_2126534.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Calcium Assay, esiRNA, Real-time Polymerase Chain Reaction, shRNA, Software, Hybridization, Modification

Journal: International journal of molecular sciences

Article Title: Modulation of Unfolded Protein Response Restores Survival and Function of β-Cells Exposed to the Endocrine Disruptor Bisphenol A.

doi: 10.3390/ijms24032023

Figure Lengend Snippet: Figure 3. Insulin processing is affected by BPA and Tm in MIN6 cells and isolated islets. (A) Time course of BPA-induced Ins2 gene expression at different time points (2, 6, 12, 24, 48 h) in MIN6 cells. RT-qPCR analysis of the Ins2 gene in MIN6 cells (B) and isolated islets (C) after 24 h of treatment with different concentrations of BPA and 5 µg/mL Tm. Fold change values were calculated by normalization to Gapdh and then to the NT values. (D) Representative fluorescence microscopy images with MIN6 cells after 24 h of treatment with 100, 250 µM BPA and 5 µg/mL Tm, marked for proinsulin (red) and insulin (green). The nucleus was stained with DAPI. (E) Graphic representation of the mean intensity of the red fluorescent channel (proinsulin) and (F) the mean intensity of the green fluorescent channel (insulin) per cell (n > 200 cells) in MIN6 cells after 24 h of treatment with increasing concentrations of BPA (as shown) and 5 µg/mL Tm. The mean intensity was determined with QuPath. Graphs display the median and the 25th and 75th percentiles. * p < 0.05, **** p < 0.0001, ns—not significant, based on one way ANOVA.

Article Snippet: Antibodies The following antibodies were used for immunofluorescence: mouse monoclonal anti-human/mouse proinsulin biotinylated antibody (1:200; R&D Systems (Minneapolis, MN, United States), #BAM13361), mouse monoclonal anti-calnexin antibody (1:100; Novus Biologicals (Englewood, CO, USA), #NB300-518), rabbit polyclonal anti-GM130 antibody (1:200; Novus Biologicals, #NBP2-53420), guinea pig polyclonal anti-insulin antibody (1:50; GeneTex (Zeeland, MI, USA), #GTX27842).

Techniques: Isolation, Gene Expression, Quantitative RT-PCR, Microscopy, Staining

Journal: Diabetes/metabolism research and reviews

Article Title: Overexpression of ZAC impairs glucose-stimulated insulin translation and secretion in clonal pancreatic beta-cells.

doi: 10.1002/dmrr.2325

Figure Lengend Snippet: Figure 6. Overexpression of ZAC inhibited proinsulin biosynthesis. (A) INS-R9-2 cells were cultured with or without doxycycline (Dox) for 48 and 96 h, and Ins2 mRNA was examined by real-time polymerase chain reaction. (B) INS-R9-2 cells were cultured with or without Dox for 96 h, cells were pulse labeled with 35S methionine/cysteine for 30 min. Equal amounts of lysate according to total protein were subjected to immunoprecipitation by proinsulin antibody and analysed by tricine-SDS-PAGE. (C) Total protein biosyn- thesis was determined by trichloroacetic acid precipitation. (D) Specific proinsulin biosynthesis was obtained through normalizing proinsulin biosynthesis in (B) by the total protein synthesis in (C). (E) Specific proinsulin biosynthesis presented as the fold induction at high glucose relative to the level at low glucose. Data represent the mean SEM of three independent experiments performed in triplicate. *p < 0.05 compared with the absence of Dox

Article Snippet: Total protein synthesis was measured by trichloroacetic acid precipitation, and lysate, normalized for total protein, was immunoprecipitated with proinsulin antibody (Novus, Littleton, CO, USA) and protein A agarose (Stratagene), and eluted in 3% SDS (wt/vol), 1.5% mercaptoethanol (vol/vol), 7.5% glycerol (wt/vol), 0.0125% Coomassie blue G-250 (Serva), 37.5 mM Tris/HCl (pH 7.0).

Techniques: Over Expression, Cell Culture, Real-time Polymerase Chain Reaction, Labeling, Immunoprecipitation, SDS Page, TCA Precipitation