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: eLife

Article Title: PDIA1/P4HB is required for efficient proinsulin maturation and ß cell health in response to diet induced obesity

doi: 10.7554/eLife.44528

Figure Lengend Snippet: ( A ) Diagram depicts the generation of Pdia1:RIP-Cre ERT mice. Mice with floxed Pdia1 alleles were crossed with RIP-Cre ERT transgenic mice and progeny were injected IP with Tam to induce Cre ERT function and Pdia1 deletion. Control littermate mice with one or two floxed Pdia1 alleles, but not harboring the RIP-Cre ERT transgene, were injected in parallel with Tam. ( B–D ) Pdia1 deletion is specific. ( B ) Total RNA was extracted from islets isolated from female mice at eight wks after Tam injection. mRNA levels were measured by qRT-PCR. Mean ± SEM, n = 3 for each group (p<0.01**). ( C ) Western blot illustrates expression of Vinculin, PDIA1, BiP, PDIA6, GRP94, Proinsulin, and Insulin in islets isolated from female mice at 14 wks after Tam injection. ( D ) Quantification of indicated proteins by Western blotting (from C ) is shown. Each value was normalized to vinculin except for the proinsulin to insulin ratio. Pdia1 fl/fl (n = 2), Pdia1 fl/+ (n = 2), Pdia1 fl/fl;Cre (n = 2).

Article Snippet: Sections were stained with the following antibodies; α-glucagon (Abcam, K79bB10), α-PDIA1 (Proteintech, 11245–1-AP), α-proinsulin (HyTest Ltd., 2PR8, CCI-17), and DAPI (Fisher Scientific).

Techniques: Transgenic Assay, Injection, Control, Isolation, Quantitative RT-PCR, Western Blot, Expressing

Journal: eLife

Article Title: PDIA1/P4HB is required for efficient proinsulin maturation and ß cell health in response to diet induced obesity

doi: 10.7554/eLife.44528

Figure Lengend Snippet: ( A–C ) Pancreas tissue sections were prepared from female mice at 49 wks after Tam injection and immuno-stained with anti- proinsulin, insulin, PDIA1, and glucagon antibodies. Images were merged with DAPI stain. Scale bar, 20 µm. ( D ) Old KO mice developed glucose intolerance compared to control genotypes measured by glucose tolerance testing (GTT) at nine wks after Tam injection. Male mice at 9 mon of age were injected with Tam and fed a regular chow. Mice were fasted (4 hr) prior to IP glucose injection (2 g/Kg body weight) and glucose levels were measured by tail bleeding at each time point (0; non-injected, 15, 30, 60, 90 min). control genotypes: Pdia1 fl/+ (n = 2), Pdia1 fl/+;Cre (n = 5), KO; n = 5. ( E ) Area under the GTT curve (Δ-AUC) of ( D ) is indicated in graph.

Article Snippet: Sections were stained with the following antibodies; α-glucagon (Abcam, K79bB10), α-PDIA1 (Proteintech, 11245–1-AP), α-proinsulin (HyTest Ltd., 2PR8, CCI-17), and DAPI (Fisher Scientific).

Techniques: Injection, Staining, Control

Journal: eLife

Article Title: PDIA1/P4HB is required for efficient proinsulin maturation and ß cell health in response to diet induced obesity

doi: 10.7554/eLife.44528

Figure Lengend Snippet: All mice were Tam injected at three wks after HFD was started. ( A ) No difference was observed in body weight (g) between control genotypes and KO mice. Mean ± SEM, controls; n = 17, KO; n = 12. ( B ) Fasting (4 hr) blood glucose levels were elevated in KO mice at 11, 16 and 20 wks after HFD. Glucose levels were measured by tail bleeding. Mean ± SEM, control genotypes; n = 17, KO; n = 12. ( C ) KO mice displayed higher blood glucose levels and area under the GTT curve (Δ-AUC) compared to control genotypes during glucose tolerance testing (GTT) after HFD for 25 wks. GTT were performed at multiple time points after HFD in two independent cohorts and representative results are shown. Mice were fasted (4 hr) prior to IP glucose injection (1 g/Kg body weight) and glucose levels were measured by tail bleeding at each time point (0; non-injected, 15, 30, 60, 90 min). control genotypes; n = 17, KO; n = 6. ( D ) KO mice exhibited decreased serum insulin levels and an increased serum proinsulin/insulin ratio compared to control genotypes. Insulin and proinsulin ELISAs were performed with the serum obtained from mice after fasting (overnight) and re-feeding (4 r) after HFD for 17 wks. control genotypes; n = 8, KO; n = 12. ( E ) No difference was observed in insulin tolerance tests performed after HFD for 20 wks. Mice were fasted for 4 hr before IP injection of insulin (1.5units/Kg). Glucose levels were measured by tail bleeding at each time point (0; non-injection, 15, 30, 60, 90, 120 min). control genotypes; n = 17, KO; n = 12. ( F–I ) Total RNA was extracted from islets isolated from mice after HFD for 30 wks. mRNA expression was measured by qRT-PCR. control genotypes; n = 3, KO; n = 3 mice. ( F ). ß cell-, α cell- and insulin processing genes. ( G ) PDI family and SERCA genes. ( H ) UPR genes. ( I ) Antioxidant response- and cell death- related genes. All data are shown as Mean ± SEM. p<0.05*, p<0.01**, p<0.001***. 10.7554/eLife.44528.007 Figure 3—source data 1. Primer sequences used for qRT-PCR.

Article Snippet: Sections were stained with the following antibodies; α-glucagon (Abcam, K79bB10), α-PDIA1 (Proteintech, 11245–1-AP), α-proinsulin (HyTest Ltd., 2PR8, CCI-17), and DAPI (Fisher Scientific).

Techniques: Injection, Control, Isolation, Expressing, Quantitative RT-PCR

Journal: eLife

Article Title: PDIA1/P4HB is required for efficient proinsulin maturation and ß cell health in response to diet induced obesity

doi: 10.7554/eLife.44528

Figure Lengend Snippet: ( A, C ) Western blotting was performed for murine islets isolated after HFD for 30 wks. After overnight recovery, islets were lysed and analyzed under reducing ( A ) or non-reducing ( C, D ) conditions by SDS-PAGE and Western blotting. Image exposed for a different time for reduced proinsulin (CCI-17) in ( A ) was used to represent the total proinsulin levels in ( D ). Six independent mice were analyzed and technical duplicates are indicated as’ on the top of gel. ( B ). Quantification of indicated proteins was performed under reducing conditions ( A ). Each value was normalized to vinculin. Proinsulin/insulin ratios were calculated based on the quantification of proinsulin and insulin species under reducing conditions ( A ). ( C ) Five samples from A were analyzed under non-reducing conditions on a 4–12% Bis-Tris SDS gel. The proinsulin blot under reducing conditions is under the red line. Left side. Quantification of HMW proinsulin complexes under non-reducing conditions is indicated. ( C ), left side, upper) The ratio of 49–198 kDa ( a ) to 14–49 kDa ( b ) proinsulin complexes is shown. ( C ), left side, lower) The ratio of 14–49 kDa ( b ) proinsulin complexes to proinsulin monomer under reducing conditions is shown. Mean value is indicated in graph. ( D ) The ten samples in A were analyzed under non-reducing conditions after the gel was incubated in 25 mM DTT for 10 min at RT prior to transfer. To control for variable transfer from a gradient gel, we used a fixed percentage gel (12% Bis-Tris SDS). Right side. Quantification is shown for HMW proinsulin complexes under non-reducing conditions. ( D ), right side, upper) The ratio of 49–198 kDa ( a ) to 14–49 kDa ( b ) proinsulin complexes is shown. ( D ), right side, lower) The ratio of 14–49 kDa ( b ) proinsulin complexes to proinsulin monomer under reducing conditions is shown. ( A–D ) genetic controls; n = 3, KO; n = 3 mice. Mean ± SEM, p<0.05*, p<0.01**, p<0.001***. ( E ) WT murine islets were treated with or without NEM and lysates were analyzed under non-reducing conditions. Equal numbers of islets were divided into two tubes. Left side islets were rinsed with cold-PBS and lysed on ice. Right side islets were rinsed with cold-PBS containing 20 mM NEM and lysed in lysis buffer containing 2 mM NEM. Samples were prepared alongside and lysates were boiled for 5 min. Equal amounts of lysates were loaded and analyzed with or without gel incubation in 25 mM DTT for 10 min at RT prior to transfer. Two different exposure time images (short, long) are shown after DTT incubation. ( F ) WT murine islets were treated with increasing concentrations of DTT for 20 min in culture at room temperature and then analyzed by non-reducing and reducing SDS-PAGE and Western blotting with proinsulin antibody (CCI-17). The range of oligomeric proinsulin species are identified by an open arrowhead and monomeric proinsulin is indicated by black arrowhead.

Article Snippet: Sections were stained with the following antibodies; α-glucagon (Abcam, K79bB10), α-PDIA1 (Proteintech, 11245–1-AP), α-proinsulin (HyTest Ltd., 2PR8, CCI-17), and DAPI (Fisher Scientific).

Techniques: Western Blot, Isolation, SDS Page, SDS-Gel, Incubation, Control, Lysis

Journal: eLife

Article Title: PDIA1/P4HB is required for efficient proinsulin maturation and ß cell health in response to diet induced obesity

doi: 10.7554/eLife.44528

Figure Lengend Snippet: WT murine islets were treated with menadione in the presence or absence of 30 μM KSC-34 for 3 hr and then treated with menadione (100μM) for 1 hr at 37°C. Five independent experiments were performed and representative results are shown. ( A ) Islet lysates were prepared and analyzed by non-reducing and reducing SDS-PAGE for Western blotting with CCI-17 antibody. ( B ) Quantification shows the proportion of HMW complexes (49–198 kDa) relative to the amount of reduced proinsulin monomer. Mean ± SEM, p<0.05*, p<0.01**, p<0.001***. Controls; n = 5, KO; n = 5 mice.

Article Snippet: Sections were stained with the following antibodies; α-glucagon (Abcam, K79bB10), α-PDIA1 (Proteintech, 11245–1-AP), α-proinsulin (HyTest Ltd., 2PR8, CCI-17), and DAPI (Fisher Scientific).

Techniques: SDS Page, Western Blot

Journal: eLife

Article Title: PDIA1/P4HB is required for efficient proinsulin maturation and ß cell health in response to diet induced obesity

doi: 10.7554/eLife.44528

Figure Lengend Snippet: ( A ) Islets were isolated from female mice at 14 wks after Tam injection, as described in , and analyzed by Western blotting under non-reducing conditions. The longer exposed image of reduced proinsulin (CCI-17) in is shown to represent the total proinsulin levels. ( B ) The same amounts of lysates from A were analyzed under non-reducing conditions after the gel was incubated in 25 mM DTT for 10 min at RT prior to transfer. To avoid unequal transfer from a gradient gel, we used a fixed percentage gel (12% Bis-Tris SDS). The proinsulin blot under reducing conditions is located under the red line. ( D ) The quantification of disulfide-linked proinsulin complexes ( B ) is shown. The left side of ( C–D ) shows the ratio of 49–198 kDa ( a ) to 14–49 kDa ( b ) proinsulin complexes. The right side of ( C–D ) shows the ratio of 14–49 kDa ( b ) proinsulin complexes to proinsulin monomer under reducing conditions.

Article Snippet: Sections were stained with the following antibodies; α-glucagon (Abcam, K79bB10), α-PDIA1 (Proteintech, 11245–1-AP), α-proinsulin (HyTest Ltd., 2PR8, CCI-17), and DAPI (Fisher Scientific).

Techniques: Isolation, Injection, Western Blot, Incubation

Journal: eLife

Article Title: PDIA1/P4HB is required for efficient proinsulin maturation and ß cell health in response to diet induced obesity

doi: 10.7554/eLife.44528

Figure Lengend Snippet: A . Islets isolated from mice after 30 wks HFD were treated with or without Menadione (10 µM, 3 hr) and co-stained with CellROX Deep Red (red) and Hoechst 33342 (blue). Live islet images were obtained by an Opera Phenix high content screening system (63X objective lens) and seven z-stack images (1 µm interval) were combined. Scale bar, 20 μm. genetic controls; n = 3, KO; n = 3. ( B ) Quantification of ROS mean intensity is shown. CellROX Deep Red mean intensity (divided by area) was measured by image J software. Mean ± SEM, p<0.001***. ( C ). Quantification of nuclear mean area (µm 2 ) measured in Hoechst 33342 stained images by ImageJ software is shown. Mean ± SEM, p<0.001***. ( D ). Histogram analysis of nuclear sizes is shown. Percent frequencies are indicated in the graph. ( E ) Western blot of islets isolated from mice after 37 wks of HFD by SDS-PAGE under reducing and non-reducing conditions is shown. After overnight recovery, islets were treated with menadione (100 µM) for 1 hr. Islet preparations from five independent control and KO mice were performed and representative images are shown. Quantification of the ratio of HMW proinsulin complexes ( a ) to monomeric proinsulin under reducing conditions is shown in graph (lower). Mean ± SEM, p<0.05*, p<0.01**, p<0.001***. Controls; n = 5, KO; n = 5 mice. ( F ) WT murine islets were treated with Menadione (100 µM) for 1 hr, treated with or without NEM as in , and lysates were prepared and analyzed under non-reducing conditions.

Article Snippet: Sections were stained with the following antibodies; α-glucagon (Abcam, K79bB10), α-PDIA1 (Proteintech, 11245–1-AP), α-proinsulin (HyTest Ltd., 2PR8, CCI-17), and DAPI (Fisher Scientific).

Techniques: Isolation, Staining, High Content Screening, Software, Western Blot, SDS Page, Control

Journal: eLife

Article Title: PDIA1/P4HB is required for efficient proinsulin maturation and ß cell health in response to diet induced obesity

doi: 10.7554/eLife.44528

Figure Lengend Snippet: After overnight recovery, WT islets were incubated in media containing brefeldin A (BFA, 5 µg/ml) and/or cycloheximide (CHX, 100 μg/ml) for 1 hr at 37°C and then analyzed by non-reducing and reducing SDS-PAGE and Western blotting with proinsulin antibody (CCI-17).

Article Snippet: Sections were stained with the following antibodies; α-glucagon (Abcam, K79bB10), α-PDIA1 (Proteintech, 11245–1-AP), α-proinsulin (HyTest Ltd., 2PR8, CCI-17), and DAPI (Fisher Scientific).

Techniques: Incubation, SDS Page, Western Blot

Journal: eLife

Article Title: PDIA1/P4HB is required for efficient proinsulin maturation and ß cell health in response to diet induced obesity

doi: 10.7554/eLife.44528

Figure Lengend Snippet: Adenoviruses that express human proinsulin (Ad-hProins) or human PDIA1 (Ad-hPDI) or catalytically inactive PDIA1 mutant (Ad-hPDImut) were infected into WT MEFs. After 48 hr, cells were treated with DTT (2.5, 5 mM) for 20 min or Menadione (50, 100 μM) for 1 hr. Lysates were analyzed by non-reducing and reducing SDS-PAGE and Western blotting with anti-human proinsulin antibody (1B24). Unfortunately, this antibody for human proinsulin does not recognize HMW proinsulin aggregates.

Article Snippet: Sections were stained with the following antibodies; α-glucagon (Abcam, K79bB10), α-PDIA1 (Proteintech, 11245–1-AP), α-proinsulin (HyTest Ltd., 2PR8, CCI-17), and DAPI (Fisher Scientific).

Techniques: Mutagenesis, Infection, SDS Page, Western Blot

Journal: eLife

Article Title: PDIA1/P4HB is required for efficient proinsulin maturation and ß cell health in response to diet induced obesity

doi: 10.7554/eLife.44528

Figure Lengend Snippet: Here, we show that PDIA1 is not required but increases the efficiency of proinsulin maturation, possibly by reducing HMW proinsulin complexes. In the absence of PDIA1, disulfide bond formation, reduction and/or isomerization in proinsulin are inadequate during increased demand so HMW proinsulin complexes accumulate in the ER upon metabolic pressure, such as a HFD. The figure depicts the formation of proinsulin HMW complexes with cellular proteins and the role of PDIA1 in interconversions to reduced proinsulin with subsequent oxidation to dimer/oligomeric proinsulin and oxidized folded proinsulin.

Article Snippet: Sections were stained with the following antibodies; α-glucagon (Abcam, K79bB10), α-PDIA1 (Proteintech, 11245–1-AP), α-proinsulin (HyTest Ltd., 2PR8, CCI-17), and DAPI (Fisher Scientific).

Techniques:

Journal: eLife

Article Title: PDIA1/P4HB is required for efficient proinsulin maturation and ß cell health in response to diet induced obesity

doi: 10.7554/eLife.44528

Figure Lengend Snippet:

Article Snippet: Sections were stained with the following antibodies; α-glucagon (Abcam, K79bB10), α-PDIA1 (Proteintech, 11245–1-AP), α-proinsulin (HyTest Ltd., 2PR8, CCI-17), and DAPI (Fisher Scientific).

Techniques: Transgenic Assay, In Vivo, Clone Assay, Produced, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: OSGEP regulates islet β-cell function by modulating proinsulin translation and maintaining ER stress homeostasis in mice

doi: 10.1038/s41467-024-54905-8

Figure Lengend Snippet: ITT assay detected the activity of insulin extracted from WT and Osgep- Ins2 - Cre mice pancreas at CD ( a ) or HFD ( b )-fed, and insulin standard as positive control ( n = 4). c AUC of ITT curve ( n = 4). d Schematic of the universal t 6 A 37 biosynthesis pathway in mammalian cell. Kinase, Endopeptidase, and Other Proteins of Small size (KEOPS) compound, with OSGEP as the active catalytic enzyme, utilizes threonylcarbamoyl adenylate (TC-AMP) to catalyze the formation of t 6 A 37 modification in ANN-decoding tRNA substrates. e HPLC-MS/MS chromatograms of t 6 A 37 in islet tRNA and relative t 6 A 37 levels were showed in WT and Osgep- Ins2 - Cre mice islet ( n = 4). f Circular dichroism spectra of protein extracted from WT and Osgep- Ins2 - Cre islet at CD. Proinsulin protein was analyzed by reducing western blots, and misfolded Proinsulin protein was analyzed by non-reducing Western blots in islets of mice under CD ( g ) and HFD ( h )-fed condition. The ratios of Monomer to total Proinsulin were calculated. The ratios of Dimers, Trimers plus High-Molecular-Weight (HMW) to total Proinsulin were calculated ( n = 3 biological replicates). Results are repeated three times independently with similar results. i Experimental procedure of in vitro transcription and translation for proinsulin protein. j Western blots analyzed the proinsulin product in addition of tRNAs from islets of HFD-fed WT and Osgep- Ins2 - Cre mice in vitro reticulocytes translation system ( n = 3). a – c , e , g , h , j Data are presented as the mean ± SD. a , b One-way ANOVA with Tukey’s post-test. c , e , g , h , j Unpaired two-tailed Student’s t-test. * p < 0.05, ** p < 0.01; *** p < 0.001; **** p < 0.0001. Source data are provided as a Source Data file. CD Chow diet, HFD High fat diet, KEOPS Kinase, Endopeptidase, and Other Proteins of Small size, TC-AMP threonylcarbamoyl adenylate, RRL Rabbit Reticulocyte Lysate.

Article Snippet: After blocking with 5% skim milk, the membrane was incubated overnight at 4 °C with one of the following primary antibodies: Osgep (SANTA CRUZ, sc-393199, 1:500 dilution in blocking buffer), β-Tubulin (Proteintech, 10094-1-AP, 1:20000 in blocking buffer), Insulin (Proteintech, 66198-1-Ig, 1:2000 in blocking buffer), Proinsulin, (Novus, NB100-73013, 1:1000 in blocking buffer), IRE1α (Cell Signaling Technology, 3294, 1:1000 in blocking buffer), Bip (Proteintech, 66574-1-Ig, 1:1000 in blocking buffer), PERK (Sigma, P0074, 1:2000 in blocking buffer), Phospho-PERK (Sigma, SAB5700521, 1:1000 in blocking buffer), eIF2α (Cell Signaling Technology, 5324, 1:2000 in blocking buffer), Phospho-eIF2α (Cell Signaling Technology, 3398, 1:1000 in blocking buffer), ATF4 (Cell Signaling Technology, 11815, 1:1000 in blocking buffer), CHOP (Cell Signaling Technology, 2895, 1:1000 in blocking buffer), ATF6 (Proteintech, 66563-1-Ig, 1:1000 in blocking buffer), AKT (Proteintech, 60203-2-Ig, 1:2000 in blocking buffer), p-Akt1/2/3 (SANTA CRUZ, SC-514032, 1:500 in blocking buffer) or Gapdh (Proteintech, 60004-1-Ig, 1:50000 in blocking buffer).

Techniques: Activity Assay, Positive Control, Modification, Tandem Mass Spectroscopy, Circular Dichroism, Western Blot, High Molecular Weight, In Vitro, Two Tailed Test

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