px330 u6 chimeric bb cbh hspcas9 expression constructs  (Addgene inc)

Journal: Nature Communications

Article Title: TBL1X/TBL1XR1 govern β-cell identity through a PAX6-containing gene regulatory network

doi: 10.1038/s41467-026-72077-5

Figure Lengend Snippet: a Scheme for the generation of an inducible β-cell-specific TBL1X and TBL1XR1 knockout. b Random fed blood glucose levels over time in iTBL/RβKO ( n = 9) and control ( n = 9) mice on HFD. c Total pancreas insulin content normalized to protein levels in iTBL/RβKO and control mice 4 and 11 weeks after tamoxifen administration. n = 3 for 4w control and iTBL/RβKO mice, n = 6 for 11w control mice, n = 4 for 11w iTBL/RβKO mice. d , e Blood glucose ( d ) and plasma insulin ( e ) levels during an oral glucose tolerance test after 22 weeks of HFD. Corresponding area under the curve in iTBL/RβKO ( n = 8) and control ( n = 9) mice ( d , right). f , g Relative mRNA expression determined by qPCR in pancreatic islets of iTBL/RβKO and control mice of Tbl1x and Tbl1xr1 ( f ) and islet genes ( g ). Control mice Tbl1x , Tbl1xr1 , Ins1 , Ins2 , Nkx6.1 , Slc2a2 , Mafa , Pdx1 , Pax6 , Ucn3 : n = 9, control mice ChgA , Ngn3 , Ldha , Hk1 : n = 8, iTBL/RβKO mice Tbl1x , Ngn3 , Ldha , Hk1 : n = 7, iTBL/RβKO mice Tbl1xr1 , Ins1 , Ins2 , Slc2a2 , Mafa n = 6, iTBL/RβKO mice Nkx6.1 , Pdx1 , Pax6 , Ucn3 , ChgA : n = 5. h α/β-cell mass ratio of pancreatic islets from iTBL/RβKO ( n = 4) and control mice ( n = 4) on HFD, 24 weeks after knockout induction. i Representative immunofluorescent staining of insulin + (blue, β-cells) and glucagon + (red, α-cells) cells of paraffin-embedded pancreas from iTBL/RβKO and control mice on HFD, 24 weeks after knockout induction. Each point represents one mouse. Data are represented as mean ± SEM. The following statistical tests were applied: two-sided student’s t test ( d – Area under the curve, f – h ) and 2-way ANOVA with Šidák’s multiple comparison post hoc test ( b – d – time course, e ). Source data are provided as a Source Data file.

Article Snippet: For this, INS1E cells were transfected with control siRNA or siRNA against TBL1X and TBL1XR1 for 72 h. After a medium change, cells were transfected with PAX6 expression constructs (purchased from Origene, BC011272 ), reporter gene constructs, and respective controls using Lipofectamine 2000 (Invitrogen, Cat# 11668027) for 48 h. For HDAC3 inhibition, expression vector transfected cells were exposed to either 10 μM BRD3308 (Sigma Aldrich, Cat# SML1639) or 5 μM RGFP966 (Cayman Chemicals, Cat# BYT-ORB636772) for 24 h. Firefly luciferase activity was normalized to renilla luciferase.

Techniques: Knock-Out, Control, Clinical Proteomics, Expressing, Staining, Comparison

Journal: Nature Communications

Article Title: TBL1X/TBL1XR1 govern β-cell identity through a PAX6-containing gene regulatory network

doi: 10.1038/s41467-026-72077-5

Figure Lengend Snippet: a , b 5 selected metabolic processes identified via gene ontology analysis using significantly enriched TBL1X ( a ) and TBL1XR1 ( b ) interaction partners with a unique peptide count > 2 identified in the interactome screen. c , Endogenous TBL1X, TBL1XR1, HDAC3, and SPT16 was immunoprecipitated in INS1E cell lysates with rabbit IgG as negative control. Whole cell lysates (Input) and immunoprecipitated proteins were immunoblotted using the indicated antibodies. d TBL1X, TBL1XR1, and vinculin protein levels in INS1E cells after siRNA transfection. e Murine Ins2 ( mIns2 ) promoter activity in INS1E cells upon TBL/R1 knockdown or/and PAX6 overexpression. Luciferase activity normalized to renilla luciferase. n = 4 wells/condition. f Endogenous HDAC3 and SPT16 was immunoprecipitated in INS1E cell lysates upon TBL/R1 knockdown with rabbit IgG as a negative control. Whole cell lysates (Input) and immunoprecipitated proteins were immunoblotted using the indicated antibodies. g Murine Ins2 ( mIns2 ) promoter activity in INS1E cells upon TBL/R1 knockdown and/or PAX6 overexpression with/ without HDAC3 inhibitor (BRD3308, 1 µM, 24 h). Luciferase activity normalized to renilla luciferase. n = 4 wells/condition. h Relative Ins2 gene expression in INS1E cells upon TBL/R1 knockdown determined by qPCR. n = 4 wells/condition. i Insulin secretion relative to insulin content in INS1E cells upon Tbl1xr1 overexpression. Insulin secretion was stimulated using 2 mM glucose (2 G), 20 mM glucose (20 G), and 2 mM glucose supplemented with KCl (KCl). Control 2 G n = 3 wells/condition, all other data n = 4 wells/condition. j Insulin secretion in murine pancreatic islets upon Tbl1xr1 overexpression. Insulin secretion was stimulated using 2.8 mM glucose (2.8 G), 16.8 mM glucose (16.8 G), and 2.8 mM glucose supplemented with KCl (KCl). n = 5. k Ins2 gene expression determined by qPCR in murine pancreatic islets upon adenovirus-mediated Tbl1xr1 overexpression. n = 3 wells/condition. Data are represented as mean ± SEM. The following statistical tests were applied: 1-way ANOVA with Dunnett’s multiple comparison post hoc test ( e ), 2-way ANOVA with a Tukey’s multiple comparison post hoc test ( g ), and a two-sided student’s t test ( h – k ). ns = no significance,* p < 0.05 ** p < 0.01, *** p < 0.001, **** p < 0.0001. Source data are provided as a Source Data file.

Article Snippet: For this, INS1E cells were transfected with control siRNA or siRNA against TBL1X and TBL1XR1 for 72 h. After a medium change, cells were transfected with PAX6 expression constructs (purchased from Origene, BC011272 ), reporter gene constructs, and respective controls using Lipofectamine 2000 (Invitrogen, Cat# 11668027) for 48 h. For HDAC3 inhibition, expression vector transfected cells were exposed to either 10 μM BRD3308 (Sigma Aldrich, Cat# SML1639) or 5 μM RGFP966 (Cayman Chemicals, Cat# BYT-ORB636772) for 24 h. Firefly luciferase activity was normalized to renilla luciferase.

Techniques: Immunoprecipitation, Negative Control, Transfection, Activity Assay, Knockdown, Over Expression, Luciferase, Gene Expression, Control, Comparison

Journal: Nature Communications

Article Title: TBL1X/TBL1XR1 govern β-cell identity through a PAX6-containing gene regulatory network

doi: 10.1038/s41467-026-72077-5

Figure Lengend Snippet: a IgG (negative control), H3K27ac (positive control), TBL1X, TBL1XR1, and PAX6 ChIP-qPCR in EndoC-βH1 cells for the human INS locus. The enrichment is calculated over the negative locus and the IgG control. n = 3 individual experiments. b Endogenous TBL1X, TBL1XR1, HDAC3, and SPT16 were immunoprecipitated in EndoC-βH1 lysates with rabbit IgG as negative control. Whole cell lysates (Input) and immunoprecipitated proteins were immunoblotted using the indicated antibodies. Representative blot is shown for 4 independent experiments. c Human INS ( hINS ) promoter activity in INS1E cells upon TBL/R1 knockdown. Luciferase activity normalized to renilla luciferase. n = 4 wells/condition. d Insulin secretion relative to insulin content in EndoC-βH1 cells upon TBL/R1 knockdown. After glucose starvation, insulin secretion was stimulated using 2.8 mM glucose (2.8 G) and 2.8 mM glucose supplemented with KCl (KCl). n = 4 wells/condition. e Scatter plot depicting the relationship between HbA1c [%] and relative TBL1X mRNA levels determined by qPCR, adjusted for age, body mass index (BMI), and disease state. Each dot represents an individual data point. n = 31. f , g Manhattan plot showing an association between elevated HbA1c levels and single nucleotide polymorphisms in the genetic region of TBL1X ( f ) and TBL1XR1 ( g ). Data are represented as mean ± SEM. The following statistical tests were applied: two-sided student’s t test ( a , c , d ), and linear regression ( e ). Source data are provided as a Source Data file.

Article Snippet: For this, INS1E cells were transfected with control siRNA or siRNA against TBL1X and TBL1XR1 for 72 h. After a medium change, cells were transfected with PAX6 expression constructs (purchased from Origene, BC011272 ), reporter gene constructs, and respective controls using Lipofectamine 2000 (Invitrogen, Cat# 11668027) for 48 h. For HDAC3 inhibition, expression vector transfected cells were exposed to either 10 μM BRD3308 (Sigma Aldrich, Cat# SML1639) or 5 μM RGFP966 (Cayman Chemicals, Cat# BYT-ORB636772) for 24 h. Firefly luciferase activity was normalized to renilla luciferase.

Techniques: Negative Control, Positive Control, ChIP-qPCR, Control, Immunoprecipitation, Activity Assay, Knockdown, Luciferase

Journal: Protein Science : A Publication of the Protein Society

Article Title: Positional scanning and computational modeling reveal determinants of legumain transpeptidase activity

doi: 10.1002/pro.70563

Figure Lengend Snippet: Design of the positional scanning assay. (a) The SFTI precursor peptide (top) is incubated with human legumain (hLEG), resulting in the formation of either a linear (L‐SFTI) or a cyclic (C‐SFTI) product. (b) A positional scanning peptide library was generated in which positions P2″, P1″, P3, P2, P1', P2', and P3' were systematically substituted with the 20 proteinogenic amino acids. (c) Reaction products were analyzed by MALDI‐ToF mass spectrometry.

Article Snippet: Briefly, LEXSY P10 cells containing the wild‐type human legumain (hLEG), or the V155G‐D160Y legumain mutant expression construct or the AtLEGβ expression construct were grown in BHI medium (Jena Bioscience) supplemented with 5 mg/mL porcine Hemin (Jena Bioscience), 50 units/mL penicillin and 50 mg/mL streptomycin (Pen‐Strep, Jena Bioscience).

Techniques: Incubation, Generated, Mass Spectrometry

Journal: Protein Science : A Publication of the Protein Society

Article Title: Positional scanning and computational modeling reveal determinants of legumain transpeptidase activity

doi: 10.1002/pro.70563

Figure Lengend Snippet: Human legumain prefers positively charged amino acids at position P2' and glycine at position P1' and P1" for efficient ligation. (a) Results of the positional peptide scanning assay using a crude peptide library of 140 variants are summarized in a heatmap. The relative amount of cyclic SFTI product formed was normalized to the peptide containing alanine at the respective position. Dark gray indicates increased cyclic product formation relative to alanine; white indicates decreased formation. (b–e) MALDI‐ToF mass spectra of selected SFTI variants after incubation with human legumain (hLEG): (b) unoptimized G 1 R…FPN 14 GLA peptide, (c) G 1 R…FG 13 N 14 GLA peptide, (d) G 1 R…FPN 14 GK 16 A peptide, and (e) G 1 R…FG 13 N 14 GK 16 A peptide. The indicated peptides were resynthesized at >95% purity. Peaks corresponding to the unprocessed SFTI precursor, the cyclic SFTI product (C‐SFTI), and the linear intermediate lacking residues G 15 LA 17 or G 15 KA 17 (L‐SFTI) are annotated. A blue star indicates a peak corresponding to a demethylated (Δ14 Da) SFTI‐GLA species, while a light‐red star marks a demethylated (Δ14 Da) c‐SFTI species. This modification likely occurs at Thr 4 or Leu 16 and is frequently observed in our MS measurements (Grassi et al., ).

Article Snippet: Briefly, LEXSY P10 cells containing the wild‐type human legumain (hLEG), or the V155G‐D160Y legumain mutant expression construct or the AtLEGβ expression construct were grown in BHI medium (Jena Bioscience) supplemented with 5 mg/mL porcine Hemin (Jena Bioscience), 50 units/mL penicillin and 50 mg/mL streptomycin (Pen‐Strep, Jena Bioscience).

Techniques: Ligation, Incubation, Modification, Mass Spectrometry

Journal: Protein Science : A Publication of the Protein Society

Article Title: Positional scanning and computational modeling reveal determinants of legumain transpeptidase activity

doi: 10.1002/pro.70563

Figure Lengend Snippet: Turnover of SFTI opt is governed by the conformational stability of legumain and does not follow the classical Michaelis–Menten binding mode. (a) MALDI‐ToF mass spectra of the SFTI opt peptide after incubation with hLEG at the indicated time points. The cyclic reaction product is labeled as C‐SFTI. (b) Relative amount of cyclized SFTI opt (C‐SFTI opt ), expressed as the percentage of total SFTI in the reaction, at increasing substrate concentrations after 2 min incubation with 60 nM or 120 nM hLEG. (c) Kinetic profile of hLEG–catalyzed SFTI cyclization. The dashed line represents the fit of the data to the Michaelis–Menten model, while the solid line represents the fit to the allosteric sigmoidal model. Curve fitting was performed using GraphPad Prism 9.3.1. (d) MALDI‐ToF mass spectrum of the G 1 …N 14 SFTI peptide after incubation with hLEG. No cyclic product formation was detected.

Article Snippet: Briefly, LEXSY P10 cells containing the wild‐type human legumain (hLEG), or the V155G‐D160Y legumain mutant expression construct or the AtLEGβ expression construct were grown in BHI medium (Jena Bioscience) supplemented with 5 mg/mL porcine Hemin (Jena Bioscience), 50 units/mL penicillin and 50 mg/mL streptomycin (Pen‐Strep, Jena Bioscience).

Techniques: Stability, Binding Assay, Incubation, Labeling, Mass Spectrometry

Journal: Protein Science : A Publication of the Protein Society

Article Title: Positional scanning and computational modeling reveal determinants of legumain transpeptidase activity

doi: 10.1002/pro.70563

Figure Lengend Snippet: Asp160 in the S2' pocket of human legumain is a key determinant of substrate specificity. (a) Crystal structure of hLEG in complex with the covalent inhibitor YVAD‐cmk (Tyr‐Val‐Ala‐Asp‐chloromethylketone) (PDB: 4AW9 ). (b–d) AlphaFold 3 models of hLEG bound to the indicated peptides: (b) FPN 14 –GL, (c) FPN 14 –GK, and (d) FGN 14 –GK. (e) MALDI‐ToF mass spectrum of the unoptimized G 1 …N 14 –GLA SFTI precursor peptide after incubation with the V155G–D160Y mutant of hLEG. The corresponding spectrum for the reaction catalyzed by wild‐type hLEG is shown in the inset. (f) MALDI‐ToF mass spectrum of the SFTI opt precursor peptide after incubation with the V155G–D160Y variant of hLEG. A blue star indicates a peak corresponding to a demethylated (Δ14 Da) SFTI–GLA species, and a red star marks a demethylated (Δ14 Da) c‐SFTI species. (g) Reaction scheme of the SFTI‐cyclisation by hLEG. Protonation of Cys189 is regulated by the P2’ residue. Positively charged P2'/P2" residues may facilitate deprotonation of Cys189, either directly or indirectly by compensating the negative electrostatic influence of the nearby Glu190. In step 1 of the transpeptidation reaction, the Cys189 Sγ is attacking the scissile peptide bond leading to the formation of a thioester intermediate. In Step 2 of the reaction the intermediate is released by the N‐terminal P1" residue.

Article Snippet: Briefly, LEXSY P10 cells containing the wild‐type human legumain (hLEG), or the V155G‐D160Y legumain mutant expression construct or the AtLEGβ expression construct were grown in BHI medium (Jena Bioscience) supplemented with 5 mg/mL porcine Hemin (Jena Bioscience), 50 units/mL penicillin and 50 mg/mL streptomycin (Pen‐Strep, Jena Bioscience).

Techniques: Incubation, Mutagenesis, Variant Assay, Residue, Docking Assay, Mass Spectrometry

Journal: Protein Science : A Publication of the Protein Society

Article Title: Positional scanning and computational modeling reveal determinants of legumain transpeptidase activity

doi: 10.1002/pro.70563

Figure Lengend Snippet: AtLEGβ prefers leucine both in position P2' and P2". (a–d) MALDI‐ToF mass spectra of selected SFTI variants after incubation with human legumain (hLEG) or AtLEGβ. (a) unoptimized G 1 R…FPN 14 GLA peptide, (b) G 1 L…FG 13 N 14 GLA peptide, (c) G 1 R…FG 13 N 14 GLA peptide, and (d) G 1 E…FG 13 N 14 GLA peptide. Peaks corresponding to the unprocessed SFTI precursor, the cyclic SFTI product (C‐SFTI), and the linear intermediate lacking residues G 15 LA 17 (L‐SFTI) are annotated.

Article Snippet: Briefly, LEXSY P10 cells containing the wild‐type human legumain (hLEG), or the V155G‐D160Y legumain mutant expression construct or the AtLEGβ expression construct were grown in BHI medium (Jena Bioscience) supplemented with 5 mg/mL porcine Hemin (Jena Bioscience), 50 units/mL penicillin and 50 mg/mL streptomycin (Pen‐Strep, Jena Bioscience).

Techniques: Incubation, Mass Spectrometry

Journal: Protein Science : A Publication of the Protein Society

Article Title: Positional scanning and computational modeling reveal determinants of legumain transpeptidase activity

doi: 10.1002/pro.70563

Figure Lengend Snippet: In silico substrate specificity screening of the prime side of human legumain reproduces its preference for basic residues at P2' and identifies P1' cysteine as a covalent inhibitor. (a) Results of the AlphaFold 3‐based in silico substrate specificity screening using the sequence of hLEG and the YVAN‐XX peptide library, summarized in a heatmap colored according to the ipTM score of the respective complexes. (b) AlphaFold 3 model of hLEG bound to the YVAN‐CR peptide, which achieved the highest ipTM score (0.96) in the in silico screen. The disulfide bond formed between the P1' cysteine of the peptide and the catalytic Cys189 of hLEG is shown. (c, d) Competition assays of hLEG with the peptides GVTN‐CR and GVTN‐GR. Each peptide (2 mM) was preincubated with the fluorogenic substrate Z‐AAN‐AMC in assay buffer (pH 5.5) containing either 2 mM DTT (c) or 20 mM DTT (d). Reactions were initiated by the addition of 2 nM hLEG, and fluorescence was recorded over 20 min at 37°C.

Article Snippet: Briefly, LEXSY P10 cells containing the wild‐type human legumain (hLEG), or the V155G‐D160Y legumain mutant expression construct or the AtLEGβ expression construct were grown in BHI medium (Jena Bioscience) supplemented with 5 mg/mL porcine Hemin (Jena Bioscience), 50 units/mL penicillin and 50 mg/mL streptomycin (Pen‐Strep, Jena Bioscience).

Techniques: In Silico, Screening, Sequencing, Fluorescence