mouse anti proinsulin  (Developmental Studies Hybridoma Bank)

Journal: EMBO Molecular Medicine

Article Title: A new form of diabetes caused by INS mutations defined by zygosity, stem cell and population data

doi: 10.1038/s44321-025-00362-9

Figure Lengend Snippet: Unrelated control (black), heterozygous R6C (HET, yellow) and isogenic corrected (HET CORR, blue) iPSCs were differentiated into β cells. ( A ) iPSC-islet lysates were analyzed by SDS-PAGE under reducing conditions, electrotransfer to nitrocellulose, and immunoblotting with anti-human proinsulin. The blot was cropped and rearranged for clarity. The left panel is reused in Fig. as it represents a shared control. ( B ) Quantification of preproinsulin to proinsulin ratio from ( A ) (unrelated control n = 3, HET CORR n = 3, HET n = 4). ( C ) iPSC-islet lysates were resolved by nonreducing 12%-NuPAGE, and the completed gel was then treated with 100 mM DTT at 60 °C for 10 min before electrotransfer to nitrocellulose and immunoblotting with anti-proinsulin. n = 2. Medium from Min6 β cells transfected with human proinsulin was used as a positive control (INS, lane next to marker, M). The blot was cropped and rearranged for clarity. The left panel is reused in Fig. as it represents the shared marker and positive control. ( D ) Proinsulin and ( E ) insulin content (ng) normalized to total protein content (μg). ( F ) Proinsulin to insulin content ratio from ( D , E ). ( D – F ) HET CORR n = 4, HET n = 7. All panels: Unpaired t -test. In box plots, the median of independent experiments is shown by a horizontal line; 25 th and 75 th percentiles are at the bottom and top of the boxes; whiskers represent the minimum and maximum values. .

Article Snippet: Mouse anti-human proinsulin B-C junction sequence KTRREAEDLQ , Abmart , Cat# B-C junction; RRID: AB_2921300.

Techniques: Control, SDS Page, Electrotransfer, Western Blot, Transfection, Positive Control, Marker

Journal: EMBO Molecular Medicine

Article Title: A new form of diabetes caused by INS mutations defined by zygosity, stem cell and population data

doi: 10.1038/s44321-025-00362-9

Figure Lengend Snippet: Unrelated control (black), homozygous R6C (HOM, pink) and isogenic corrected (HOM CORR, blue) iPSCs were differentiated into β cells. ( A ) iPSC-islet lysates were analyzed by SDS-PAGE under reducing conditions, electrotransferred to nitrocellulose, and immunoblotted with anti-human proinsulin. The blot was cropped and rearranged for clarity. The left panel is reused as in Fig. , as it represents a shared control. ( B ) Quantification of preproinsulin to proinsulin ratio from ( A ) (unrelated control n = 3, HOM CORR n = 3, HOM n = 8) and ( C ) of homozygous R6C iPSC-β cells treated with vehicle DMSO or MG132 (10 μM) for 30 min ( n = 4). ( D ) iPSC-islet lysates were resolved by nonreducing 12%-NuPAGE and the completed gel then treated with 100 mM DTT at 60 °C for 10 min before electrotransfer to nitrocellulose and immunoblotting with anti-proinsulin. n = 2. Medium from Min6 β cells transfected with human proinsulin was used as a positive control (INS, lane next to marker, M). The blot was cropped and rearranged for clarity. The left panel is reused as in Fig. as it represents a shared marker and a positive control. ( E ) Proinsulin and ( F ) insulin content normalized to total protein content. ( G ) Proinsulin to insulin content ratio from ( E , F ). ( E – G ) HOM CORR n = 8, HOM n = 12–13. ( H ) Insulin content normalized to total protein content along stage 7 (S7, HOM CORR n = 5, HOM n = 12), 1 week (LC1W, HOM CORR n = 3, HOM n = 3), 2 weeks (LC2W, HOM CORR n = 3, HOM n = 3), 3 weeks (LC3W, HOM CORR n = 3, HOM n = 3), and 4 weeks (LC4W) of long culture (LC, HOM CORR n = 12, HOM n = 15). All panels: Unpaired t -test. In box plots, the median of independent experiments is shown by a horizontal line; 25 th and 75 th percentiles are at the bottom and top of the boxes; whiskers represent the minimum and maximum values. .

Article Snippet: Mouse anti-human proinsulin B-C junction sequence KTRREAEDLQ , Abmart , Cat# B-C junction; RRID: AB_2921300.

Techniques: Control, SDS Page, Electrotransfer, Western Blot, Transfection, Positive Control, Marker

Journal: EMBO Molecular Medicine

Article Title: A new form of diabetes caused by INS mutations defined by zygosity, stem cell and population data

doi: 10.1038/s44321-025-00362-9

Figure Lengend Snippet: Heterozygous R6C (HET, yellow) and isogenic corrected (HET CORR, blue) iPSCs were differentiated into long-cultured β cells. ( A – C ) Static proinsulin and insulin secretion in response to 2.8 mM glucose (G2.8), 16.8 mM glucose (G16.8), or 16.8 mM glucose plus 10 μM forskolin (G16.8 + Fk). HET CORR n = 4, HET n = 7. ( A ) Proinsulin and ( B ) insulin secretion (ng) normalized to protein content (μg). ( C ) Proinsulin to insulin ratio from ( A , B ). ( D – H ) Dynamic insulin secretion upon perifusion with 2.8 mM glucose, 16.8 mM glucose (G16.8), G16.8 plus exendin-4 (Ex4, 50 ng/mL, 11.8 nM) or G2.8 plus KCl (30 mM). HET CORR n = 3, HET n = 4. ( D ) Insulin secretion normalized to protein content, with ( E – H ) area under the curve (AUC) per minute of secretion at G2.8, G16.8, G16.8 + Ex4, and G2.8 + KCl. All panels: Unpaired t -test. In box plots, the median of independent experiments is shown by a horizontal line; 25 th and 75 th percentiles are at the bottom and top of the boxes; whiskers represent the minimum and maximum values. In time course line plots, data are shown as mean ± s.e.m. .

Article Snippet: Mouse anti-human proinsulin B-C junction sequence KTRREAEDLQ , Abmart , Cat# B-C junction; RRID: AB_2921300.

Techniques: Cell Culture

Journal: EMBO Molecular Medicine

Article Title: A new form of diabetes caused by INS mutations defined by zygosity, stem cell and population data

doi: 10.1038/s44321-025-00362-9

Figure Lengend Snippet: Homozygous R6C (HOM, pink) and isogenic corrected (HOM CORR, blue) iPSCs were differentiated into long-cultured β cells. ( A – C ) Static proinsulin and insulin secretion in response to 2.8 mM glucose (G2.8), 16.8 mM glucose (G16.8), or 16.8 mM glucose plus 10 μM forskolin (G16.8 + Fk). HOM CORR n = 8, HOM n = 12 (for proinsulin), HOM n = 13 (for insulin). ( A ) Proinsulin and ( B ) insulin secretion normalized to protein content. ( C ) Proinsulin to insulin ratio from ( A , B ). ( D – I ) Dynamic insulin secretion upon perifusion with 2.8 mM glucose, 16.8 mM glucose (G16.8), G16.8 plus exendin-4 (Ex4, 50 ng/mL, 11.8 nM), or G2.8 plus KCl (30 mM). HOM CORR n = 12, HOM n = 14. ( D ) Insulin secretion normalized to protein content, with ( E ) zoom in on 16.8 mM glucose response. ( F – I ) Area under the curve (AUC) per minute of secretion at G2.8, G16.8, G16.8 + Ex4 and G2.8 + KCl. All panels: Unpaired t -test. In box plots, the median of independent experiments is shown by a horizontal line; 25 th and 75 th percentiles are at the bottom and top of the boxes; whiskers represent the minimum and maximum values. In time course line plots, data are shown as mean ± s.e.m. .

Article Snippet: Mouse anti-human proinsulin B-C junction sequence KTRREAEDLQ , Abmart , Cat# B-C junction; RRID: AB_2921300.

Techniques: Cell Culture

Journal: Molecular Metabolism

Article Title: Beta-cell-specific C3 deficiency exacerbates metabolic dysregulation and insulin resistance in obesity

doi: 10.1016/j.molmet.2025.102302

Figure Lengend Snippet: A) Pancreas sections from treated mice were stained for glucagon (green) and insulin (red) to visualize islets and assess their size. Blue: DAPI. Scale bars, 50 μm. B) Quantification of total islet size in sections from 22 islets from pancreas sections of 7 LFD beta-C3-KO mice, 33 islets from pancreas sections of 5 HFD beta-C3-KO mice, and 35 islets from pancreas sections of 6 HFD C3-flox mice. C) Fraction of total islet staining for insulin, from the same islets analysed in (B). D) Examples of staining of individual HFD islets for glucagon (green) and pro-insulin (red). E) Quantification of pro-insulin staining in islets from each group of mice. F) Quantification of pro-insulin in serum of mice at given time points, measured by pro-insulin specific ELISA. G) Transmission electron micrographs of pancreatic islet cells isolated from beta-C3-KO or C3-flox mice. Beta-C3-KO mouse beta-cells contain accumulated amounts of swollen ER or similar vesicular organelles (black arrows), while normal ER is apparent in C3-flox mouse islet cells (white arrows). See also . Increased amounts of autophagic material were also observed in beta-C3-KO cells (asterisk). H) In vitro insulin secretion from isolated islets from C3-flox or beta-C3-KO mice HFD-fed mice, at 15 or 60 min. I) Average insulin content values per islet from secretion experiment in panel (H) (average values shown for each of 6 mice: from 6 wells, 5 islets per well). J) Serum C3 levels in C3-flox or beta-C3-KO mice at the endpoint of HFD, as measured by ELISA. Statistics in E, one-way ANOVA, in F/H, two-way ANOVA, in I, Student’s T-test, and in B/C, by mixed model statistical analysis (see methods).

Article Snippet: For pro-insulin staining, AlexaFluor647-labeled anti-pro-insulin (R&D Systems, #IC13361R, 1:100) was incubated overnight at 4 °C, in combination with glucagon staining, before washing and mounting as described above.

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Transmission Assay, Isolation, In Vitro

Journal: bioRxiv

Article Title: Loss of brain insulin production impairs learning and memory in female mice

doi: 10.1101/2025.08.15.670639

Figure Lengend Snippet: (A) Immunoreactivity for C-peptide, a product of insulin biosynthesis, is detected in the hilus, stratum lucidum (SL), and CA3 regions of hippocampus. C-peptide immunoreactive cells were observed in the granule cell layer of the dentate gyrus, as indicated by arrows. (B) Immunoblot analysis of proinsulin expression in the pancreas and hippocampus from WT and Ins2 KO mice. (C) Proinsulin-positive cells were detected and co-localized with β-gal in the CA3 regions. β-galactosidase (β-gal) staining was detected in Ins2 knockout and heterozygous mice but not wildtype (WT). Pro-insulin was detected in WT and β-gal heterozygous mice but not knockouts. (D) GFP fluorescence in hippocampal neuron cultures of from Ins2 GFP/GFP knock-in, replacement mice.

Article Snippet: The membranes were blocked with 5% skim milk for 1 hour and then incubated with primary antibody against proinsulin (1:1000; Cell Signaling Technology, 8138) or GAPDH (1:10,000; Cell Signaling Technology, 2118) at 4°C overnight.

Techniques: Western Blot, Expressing, Staining, Knock-Out, Fluorescence, Knock-In