bigbac kit  (Addgene inc)

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

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: medRxiv

Article Title: Gene-gene interactions protect against Familial Hypercholesterolemia: effect of lost- of-function PCSK9 variants

doi: 10.64898/2026.03.26.26348145

Figure Lengend Snippet: Pedigree of the family showing segregation of LDLR and PCSK9 variants. Squares represent males, circles are females and diamond symbols are used to represent family members without specifying their sex. Individuals fulfilling the clinical diagnosis criteria for familial hypercholesterolemia (FH) are indicated by half-filled symbols. The first row below each symbol correspond to the individual ID following by the genotype at LDLR, PCSK9 and APOB ..

Article Snippet: Human LDLR cDNA constructs were generated using a GFP-tagged plasmid vector (C-GFPSpark®, NM_000527; Sino Biological, Beijing, China).

Techniques: Biomarker Discovery

Journal: medRxiv

Article Title: Gene-gene interactions protect against Familial Hypercholesterolemia: effect of lost- of-function PCSK9 variants

doi: 10.64898/2026.03.26.26348145

Figure Lengend Snippet: Functional characterization of LDLR c.2479G>A ; (A) Flow cytometry histogram overlay showing DiI-LDL binding; (B) Quantification of LDL uptake relative to WT; (C) LDLR expression. The upper line represents the benignity cutoff (90%) established by the ACMG/ClinGen guidelines for functional studies. * <0.05; **** <0.0001.

Article Snippet: Human LDLR cDNA constructs were generated using a GFP-tagged plasmid vector (C-GFPSpark®, NM_000527; Sino Biological, Beijing, China).

Techniques: Functional Assay, Flow Cytometry, Binding Assay, Expressing

Journal: Nucleic Acids Research

Article Title: Scaffolding of the H4K5ac chromatin remodeling complex by lncRNA MAHAC mediates epithelial-mesenchymal transition

doi: 10.1093/nar/gkag101

Figure Lengend Snippet: MAHAC scaffolds the ILF3/NF90–ILF2–CBP complex. ( A ) Co-immunoprecipitation experiments using anti-ILF3 antibodies pulled down ILF3, NF90, ILF2, and CBP from H1299 cell extracts, whereas other HAT members (Tip60, HBO1, HAT1, p300) were not detected in the immunoprecipitates by Western blot analysis. ( B ) RNA pull-down assays confirmed the scaffolding of ILF3/NF90-ILF2-CBP complex by MAHAC using H1299 cell extracts. Biotinylated MAHAC RNA samples (sense and anti-sense) were detected by Streptavidin-HRP. Biotinylated anti-sense MAHAC and beads were used as controls. ( C ) UV-crosslinking RNA pull-down assays confirmed the endogenous interaction between MAHAC and the ILF3/NF90-ILF2-CBP complex in H1299 cell lysates. RNase A treatment was used to confirm that the observed associations were mediated by MAHAC RNA. ( D ) CLIP assays confirmed that the antibodies against ILF3/NF90, ILF2, or CBP could pull down endogenous MAHAC . UV-crosslinked cell lysates were immunoprecipitated with the indicated antibodies. The amount of co-immunoprecipitated MAHAC was quantified by RT-qPCR and expressed as a percentage of the total input RNA. ( E ) Knockdown of MAHAC abolished the interaction between CBP and ILF3/NF90-ILF2 using co-immunoprecipitation experiments of H1299 cell extracts. ( F ) RNase treatment abolished the interaction between CBP and ILF3/NF90-ILF2ILF3 using co-immunoprecipitation experiments of H1299 cell extracts. ( G ) Knockdown of MAHAC abolished the interaction between CBP and ILF3/NF90-ILF2 using co-immunoprecipitation experiments of H1299 cell extracts. Scr-si, scrambled siRNA used as a control; N, normoxia; H, hypoxia. *, P < 0.05.

Article Snippet: Human ILF3, NF90, and ILF2 expression constructs were purchased from Origene Technologies (Rockville, MD, USA).

Techniques: Immunoprecipitation, Western Blot, Scaffolding, Quantitative RT-PCR, Knockdown, Control

Journal: Nucleic Acids Research

Article Title: Scaffolding of the H4K5ac chromatin remodeling complex by lncRNA MAHAC mediates epithelial-mesenchymal transition

doi: 10.1093/nar/gkag101

Figure Lengend Snippet: Mapping of the binding regions between MAHAC and the ILF3/NF90–ILF2–CBP complex. ( A ) The predicted secondary structure of MAHAC using the RNA secondary structure prediction program. ( B ) Deletion and truncation mapping experiments showed that the nucleotides 498–1022 region in MAHAC was responsible for anchoring the ILF3/NF90–ILF2–CBP complex. ( C ) Further domain mapping experiments showed that the MAHAC ribonucleotides 658–767 region anchored the ILF3/NF90–ILF2–CBP complex. ( D ) Mapping of the region inside MAHAC showed that the region II-1 (nt 686–741) bound to the ILF3/NF90–ILF2–CBP complex. The illustration on the left shows the RNA corresponding to different fragments of the MAHAC II. Denaturing agarose gel stained with EtBr showing in vitro transcribed MAHAC II, MAHAC II-1, and MAHAC II-2 transcripts. ( E ) Mapping of the MAHAC region required for CBP interaction. Schematic illustration of the RNA secondary structures for MAHAC fragments II-1 and II-1–7. RNA pull-down assays using in vitro transcribed biotinylated MAHAC fragments. Denaturing agarose gel electrophoresis stained with EtBr confirmed the size of the in vitro transcribed MAHAC II-1 and MAHAC II-1–7 transcripts. ( F ) Overexpression of the MAHAC II-1 region induced the global H4K5ac levels.

Article Snippet: Human ILF3, NF90, and ILF2 expression constructs were purchased from Origene Technologies (Rockville, MD, USA).

Techniques: Binding Assay, Agarose Gel Electrophoresis, Staining, In Vitro, Over Expression

Journal: Nucleic Acids Research

Article Title: Scaffolding of the H4K5ac chromatin remodeling complex by lncRNA MAHAC mediates epithelial-mesenchymal transition

doi: 10.1093/nar/gkag101

Figure Lengend Snippet: The MAHAC nt 686–741 fragment is essential for its functions. ( A ) UV-crosslinking RNA pull-down assays showed that restoration with wild-type MAHAC , but not the MAHAC -Δ686–741 mutant, pulled down the ILF3/NF90-ILF2-CBP complex in H1299 cells under MAHAC knockdown. ( B ) CLIP-qPCR analysis showed that wild-type MAHAC , but not MAHAC -Δ686–741, could be pulled down by antibodies against ILF3/NF90, ILF2, or CBP antibodies in MAHAC -knockdown H1299 cells, followed by restoration with wild-type MAHAC or MAHAC -Δ686–741. ( C ) Co-immunoprecipitation experiments in MAHAC -knockdown H1299 cells showed that the restoration with wild-type MAHAC , but not MAHAC -Δ686–741, promoted the interaction between CBP and the ILF3/NF90-ILF2 complex. ( D ) The restoration with wild-type MAHAC , but not MAHAC -Δ686–741, induced H4K5ac levels in MAHAC -knockdown H1299 cells. ( E and F ) The restoration with wild-type MAHAC , but not MAHAC -Δ686–741, promoted EMT, cell migration, and invasion in MAHAC -knockdown H1299 cells. * P < 0.05.

Article Snippet: Human ILF3, NF90, and ILF2 expression constructs were purchased from Origene Technologies (Rockville, MD, USA).

Techniques: Mutagenesis, Knockdown, Immunoprecipitation, Migration

Journal: Nucleic Acids Research

Article Title: Scaffolding of the H4K5ac chromatin remodeling complex by lncRNA MAHAC mediates epithelial-mesenchymal transition

doi: 10.1093/nar/gkag101

Figure Lengend Snippet: The binding pattern between MAHAC and ILF3/NF90. ( A ) Measurement of stoichiometry showed a ratio of 1:1 between NF90 and MAHAC . The RNA EMSA data showed that the binding of NF90 to MAHAC establishes a 1:1 stoichiometry of the complex. The molar ratio of NF90 to MAHAC is listed above the lane. The positions of NF90- MAHAC complexes and free MAHAC are shown. ( B ) The stoichiometry of MAHAC bound to NF90 was measured by filter binding assay. Compare the data with theoretical saturation curves for protein:RNA stoichiometric ratios of 1:1, 2:1, and 4:1. The 1:1 curve is the closest to the data, thus confirming that one copy of MAHAC interacted with a single NF90 molecule. ( C ) Cooperative binding between ILF3 and NF90 using tagged full-length ILF3 and truncated NF90 showed that heterodimers of ILF3 and NF90 had the strongest binding to the MAHAC -II-1 region using RNA EMSA assay (lower panel). The diagram shows the protein domain of full-length ILF3 and truncated NF90 (upper panel). The usage of HA antibody (HA Ab labeling) was to supershift the ILF3 and NF90 complex. ( D ) Allosteric activation of H4K5ac by incubating MAHAC -II-1 with ILF3, NF90, ILF2, CBP, and histone H4 in in vitro HAT assays.

Article Snippet: Human ILF3, NF90, and ILF2 expression constructs were purchased from Origene Technologies (Rockville, MD, USA).

Techniques: Binding Assay, Filter-binding Assay, Labeling, Activation Assay, In Vitro

Journal: Nucleic Acids Research

Article Title: Scaffolding of the H4K5ac chromatin remodeling complex by lncRNA MAHAC mediates epithelial-mesenchymal transition

doi: 10.1093/nar/gkag101

Figure Lengend Snippet: Bioinformatics analysis of H4K5ac-marked genes regulated by hypoxia and MAHAC overexpression and a model to depict the regulation of H4K5ac mark and hypoxia-induced EMT/tumor progression by MAHAC II-1. ( A ) The pie chart showed the genome-wide distribution of H4K5ac ChIP-seq peaks under hypoxia. A total of 82204 peaks (32.08%) exhibited significant increases in H4K5ac signal under hypoxia. ( B ) Genomic annotation showed that hypoxia-induced H4K5ac peaks were significantly enriched at promoter regions compared with peaks located in other genomic regions (χ² test, P < 2 × 10 -26 ). ( C ) A Venn diagram illustrates the number of genes whose promoters were occupied by hypoxia-induced H4K5ac and regulated by hypoxia and MAHAC overexpression. ( D ) KEGG pathway analysis was performed on the 309 overlapping genes. ( E ) Functional categorization of the 309 overlapping genes revealed the biologically relevant processes that they belong to, including angiogenesis, stemness, and cancer metabolism. ( F ) A model of MAHAC- II-1 anchoring the ILF3/NF90-ILF2-CBP complex to mediate H4K5ac and promote EMT/tumor progression is depicted. The UGA-like box (shaded in pink) is shown to be the putative binding region by ILF3/NF90. A putative CBP-interacting region (shaded in yellow) within the same MAHAC- II-1 sequence is also indicated. Black color indicates lncRNA; orange color indicates histones; blue color indicates histone H4K5 acetylation.

Article Snippet: Human ILF3, NF90, and ILF2 expression constructs were purchased from Origene Technologies (Rockville, MD, USA).

Techniques: Over Expression, Genome Wide, ChIP-sequencing, Functional Assay, Binding Assay, Sequencing