mouse anti tfiib  (Santa Cruz Biotechnology)

Journal: bioRxiv

Article Title: Real-time visualization of reconstituted transcription reveals RNA polymerase II activation mechanisms at single promoters

doi: 10.1101/2025.01.06.631569

Figure Lengend Snippet: TFIIE and TFIIH increase transcription; TFIID accelerates RNAPII activation (A) Number of active promoters based upon PIC composition. All lanes include minimal system of TBP, TFIIB, TFIIF, and RNAPII; other PIC factors added as noted. If TFIID was added, TBP was not included in addition. Bars represent mean ± SEM. (B) Total transcription over time from PICs ±Mediator and/or TFIID. Asterisks signify burst duration, line depicts mean values, and shading represents SEM. No TFIID experiments included TBP instead. (C) Time to first round transcription from Full PIC (left) or PICs lacking TFIID or Mediator and TFIID. If no TFIID, TBP was used. Black bars represent mean ± SEM. (D) Burst size as function of Mediator and TFIID. Values listed are mean burst sizes from multi-round promoters (±SEM); values in brackets are mean burst size of all active promoters (±SEM). (E) Time to re-initiation with Full PIC (left) or PICs lacking TFIID or Mediator and TFIID. If no TFIID, TBP was used. Black bars represent mean ± SEM. (F) Fastest re-initiation events (n=20). Black bars represent mean ± SEM. All panels shown (A-F) represent data from 2 biological replicates.

Article Snippet: The remaining GTFs were purchased: TFIIA (Protein One #P1032-01), TBP (Active Motif #81114), TFIIB (Novus Biologicals #NBP1-30313).

Techniques: Activation Assay

Journal: The Journal of pathology

Article Title: Runt-related transcription factor 1 (RUNX1) is a mediator of acute kidney injury.

doi: 10.1002/path.6355

Figure Lengend Snippet: Figure 2. TWEAK induces RUNX1 expression and RUNX1-mediated IL-6 expression in cultured tubule cells. (A) Heatmap of Runx family genes in transcriptomics array of MCT cells stimulated with TWEAK (100 ng/ml) or vehicle for 6 h (n = 3 per group). Each column contains the raw data of gene expression for a single sample. (B) Runx1 mRNA expression assessed by RT-qPCR at 3, 6, and 24 h of stimulation with 100 ng/ml TWEAK in MCT cells. (C) Quantification and representative western blot of RUNX1 in total protein extracts from MCT cells at 3, 6, and 24 h of TWEAK stimulation. (D) Quantification and representative western blot of RUNX1 in nuclear extracts from MCT cells stimulated with TWEAK. (E) RUNX1 protein assessed by western blotting in mouse kidneys 48 h after TWEAK administration. Mean ± SD of 3–5 animals per group. *p < 0.05. (F, G) MCT cells were pretreated with RUNX1 inhibitor Ro5-3335 for 1 h before stimulation with 100 ng/ml TWEAK. Il6 mRNA was assessed by RT-qPCR at 6 h of TWEAK stimulation (F), and IL-6 released to cell supernatants was measured by ELISA at 24 h of TWEAK stimulation (G). (H) Il6 mRNA levels assessed by RT-qPCR in MCT cells transfected with RUNX1 or control siRNA and stimulated with 100 ng/ml TWEAK for 6 h. (I) ChIP assay of RUNX1 binding to the Il6 gene promoter using primers for four regions containing RUNX1-binding sites indicated on the X-axis (R1, R2, R3, R4). Results are presented as per cent enrichment over input for binding of anti-RUNX1 antibody or anti-IgG to the Il6 gene promoter in MCT cells stimulated with TWEAK for 6 h as compared with control cells. B–D and F–I show mean ± SD of 3–5 independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: Blots were washed with PBS/Tween, incubated with appropriate horseradish peroxidaseconjugated secondary antibody (1:5,000; GE Healthcare, Chicago, IL, USA), and signals were developed using a chemiluminescence method (ECL) (Millipore-Merck) and probed with mouse monoclonal anti-GAPDH (1:5,000, MAB374, Millipore-Merck) or mouse monoclonal antiα-TFIIb (1:1,000, 4169; Cell Signaling Technology, Danvers,MA, USA) antibodies.

Techniques: Expressing, Cell Culture, Gene Expression, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Transfection, Control, Binding Assay

Journal: The Journal of pathology

Article Title: Runt-related transcription factor 1 (RUNX1) is a mediator of acute kidney injury.

doi: 10.1002/path.6355

Figure Lengend Snippet: Figure 3. LPS induces RUNX1 expression and RUNX1-mediated IL-6 expression in cultured tubule cells. (A) Runx1 mRNA expression assessed by RT-qPCR after stimulation with 100 ng/ml LPS in MCT cells. (B, C) Quantification and representative western blot of RUNX1 in total protein extracts (B), and in nuclear extracts (C), from MCT cells after LPS stimulation. (D, E) MCT cells were pretreated with the RUNX1 inhibitor Ro5-3335 for 1 h before stimulation with 100 ng/ml LPS. Il6 mRNA levels were assessed by RT-qPCR at 6 h (D), and IL-6 released to cell supernatants was measured by ELISA at 24 h after LPS addition (E). (F) Il6 mRNA levels assessed by RT-qPCR in MCT cells transfected with RUNX1 or control siRNA and stimulated with 100 ng/ml LPS for 6 h. (G) ChIP assay of RUNX1 binding to the Il6 gene promoter using primers in four regions containing RUNX1-binding sites indicated on the X-axis (R1, R2, R3, R4). Results are presented as per cent enrichment over input for binding of anti-RUNX1 antibody or anti-IgG to the Il6 gene promoter in MCT cells stimulated with LPS for 6 h as compared with control cells. A–G show mean ± SD of three or four independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: Blots were washed with PBS/Tween, incubated with appropriate horseradish peroxidaseconjugated secondary antibody (1:5,000; GE Healthcare, Chicago, IL, USA), and signals were developed using a chemiluminescence method (ECL) (Millipore-Merck) and probed with mouse monoclonal anti-GAPDH (1:5,000, MAB374, Millipore-Merck) or mouse monoclonal antiα-TFIIb (1:1,000, 4169; Cell Signaling Technology, Danvers,MA, USA) antibodies.

Techniques: Expressing, Cell Culture, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Transfection, Control, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: 8-Prenylgenistein Isoflavone in Cheonggukjang Acts as a Novel AMPK Activator Attenuating Hepatic Steatosis by Enhancing the SIRT1-Mediated Pathway

doi: 10.3390/ijms25179730

Figure Lengend Snippet: Effect of 8PG on AMPK activation and SIRT1-mediated lipogenesis and fatty acid oxidation in palmitate-treated HepG2 cells. ( A ) Samples with the highest AMPK expression and samples with the lowest AMPK expression were evaluated to determine the ACC levels in the liver tissue of healthy participants (n = 215) using (GENT2) ( http://gent2.appex.kr/gent2/ , accessed on 18 January 2024) databases. The data are expressed as mean ± standard deviation (SD), and asterisks (*) indicate significant differences between groups (*** p < 0.001). ( B ) Protein expression levels of phosphorylated AMPK, and ACC, in palmitate (500 µM)-treated HepG2 cells were analyzed using Western blotting. ( C , D ) mRNA expression of SREBP-1 and ACOX1 in the liver tissue of ND- and HFD-fed mice were obtained from the GSE32095 dataset (ND, n = 3; HFD, n = 3). Asterisks (*) indicate significant differences (*** p < 0.001) compared with the normal diet group. ( E ) mRNA expression of SIRT1-linked hepatic lipogenesis and fatty acid oxidation genes in the liver of control and SIRT1 knockout mice were obtained from the GSE14921 dataset (Control, n = 3; SIRT1 ko, n = 3). Asterisks (*) indicate significant differences (* p < 0.05 and ** p < 0.01) compared with the control group. ( F ) Protein expression levels of, ACOX1, CPT1α, and nuclear expression levels of SIRT1 in palmitate (500 µM)-treated HepG2 cells were analyzed using Western blotting. TFIIB was used as a loading control for the nuclear lysates. 8PG, 8-prenylgenistein; ACC, acetyl-CoA carboxylase; ACOX1, acyl-CoA oxidase-1; AMPK, adenosine 5′ monophosphate-activated protein kinase; CPT1α, carnitine palmitoyltransferase I alpha; FAS, fatty Acid Synthase; HFD, high-fat diet, ND, normal diet, SD, standard deviation; SIRT1, sirtuin 1; SREBP-1, sterol regulatory element-binding protein 1, TCGA, The Cancer Genome Atlas Program, TFIIB, transcription factor II b.

Article Snippet: Primary antibodies against the following proteins were used for Western blot analysis: AMPK, p-AMPK (Thr172), ACC, p-ACC (Ser79), CPT1α, SIRT1, TFIIB, and β-actin; these antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Activation Assay, Expressing, Standard Deviation, Western Blot, Control, Knock-Out, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: 8-Prenylgenistein Isoflavone in Cheonggukjang Acts as a Novel AMPK Activator Attenuating Hepatic Steatosis by Enhancing the SIRT1-Mediated Pathway

doi: 10.3390/ijms25179730

Figure Lengend Snippet: Identification, functional evaluation, and network pharmacology analysis of 8PG. ( A ) Methodology for preparing cheonggukjang (CGJ). ( B , C ) Enriched KEGG pathways and biological process of the overlapped targets of 8PG based on a ShinyGO (ver0.77) web server. ( D ) Target networks in 8PG. 8PG, 8-prenylgenistein; KEGG, Kyoto encyclopedia of genes and genomes; SIRT1, sirtuin 1.

Article Snippet: Primary antibodies against the following proteins were used for Western blot analysis: AMPK, p-AMPK (Thr172), ACC, p-ACC (Ser79), CPT1α, SIRT1, TFIIB, and β-actin; these antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Functional Assay

Journal: International Journal of Molecular Sciences

Article Title: 8-Prenylgenistein Isoflavone in Cheonggukjang Acts as a Novel AMPK Activator Attenuating Hepatic Steatosis by Enhancing the SIRT1-Mediated Pathway

doi: 10.3390/ijms25179730

Figure Lengend Snippet: Molecular docking analysis of 8PG and resveratrol with SIRT1 using AutoDock 4.2.6. ( A ) Binding interactions of the control, resveratrol (pink), and 8PG (purple) with the SIRT1 protein; ( B ) Two-dimensional pharmacophore analysis between SIRT1 and the control compound, resveratrol (pink box); ( C ) Two-dimensional pharmacophore analysis between SIRT1 and the active component, 8PG (purple box). The AutoDock 4 result was used for visualization and pharmacophore analysis. Commonly shared residues between resveratrol and SIRT1 and 8PG and SIRT1, were rounded by the cyan blue dotted circle. ( D – G ) Molecular dynamics simulation of 8PG and resveratrol with SIRT1. ( D ) RMSD plots between 8PG (purple) and SIRT1 and resveratrol (red) and SIRT1. ( E ) RMSF plots between 8PG and SIRT1 and resveratrol and SIRT1. ( F ) Binding interaction plots between 8PG and resveratrol with SIRT1. ( G ) Number of hydrogen bonds between 8PG and SIRT1 and resveratrol and SIRT1 at 10 ns. SIRT1, sirtuin 1; 2D, two-dimensional; 8PG, 8-prenylgenistein; ns, nanosecond; RMSD, root-mean-square deviation; RMSF, root-mean-square fluctuation.

Article Snippet: Primary antibodies against the following proteins were used for Western blot analysis: AMPK, p-AMPK (Thr172), ACC, p-ACC (Ser79), CPT1α, SIRT1, TFIIB, and β-actin; these antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Binding Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: 8-Prenylgenistein Isoflavone in Cheonggukjang Acts as a Novel AMPK Activator Attenuating Hepatic Steatosis by Enhancing the SIRT1-Mediated Pathway

doi: 10.3390/ijms25179730

Figure Lengend Snippet: In silico docking simulation of the active metabolite, 8PG, from fermented soybean CGJ with SIRT1.

Article Snippet: Primary antibodies against the following proteins were used for Western blot analysis: AMPK, p-AMPK (Thr172), ACC, p-ACC (Ser79), CPT1α, SIRT1, TFIIB, and β-actin; these antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: In Silico, Control

Journal: International Journal of Molecular Sciences

Article Title: 8-Prenylgenistein Isoflavone in Cheonggukjang Acts as a Novel AMPK Activator Attenuating Hepatic Steatosis by Enhancing the SIRT1-Mediated Pathway

doi: 10.3390/ijms25179730

Figure Lengend Snippet: Detailed comparison of the ADMET properties of 8PG with those of the control compounds for AMPK and SIRT1.

Article Snippet: Primary antibodies against the following proteins were used for Western blot analysis: AMPK, p-AMPK (Thr172), ACC, p-ACC (Ser79), CPT1α, SIRT1, TFIIB, and β-actin; these antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Comparison, Control, Permeability, Clinical Proteomics, Protein Binding, Inhibition, Ames Test

Journal: International Journal of Molecular Sciences

Article Title: 8-Prenylgenistein Isoflavone in Cheonggukjang Acts as a Novel AMPK Activator Attenuating Hepatic Steatosis by Enhancing the SIRT1-Mediated Pathway

doi: 10.3390/ijms25179730

Figure Lengend Snippet: Effect of 8PG on AMPK activation and SIRT1-mediated lipogenesis and fatty acid oxidation in palmitate-treated HepG2 cells. ( A ) Samples with the highest AMPK expression and samples with the lowest AMPK expression were evaluated to determine the ACC levels in the liver tissue of healthy participants (n = 215) using (GENT2) ( http://gent2.appex.kr/gent2/ , accessed on 18 January 2024) databases. The data are expressed as mean ± standard deviation (SD), and asterisks (*) indicate significant differences between groups (*** p < 0.001). ( B ) Protein expression levels of phosphorylated AMPK, and ACC, in palmitate (500 µM)-treated HepG2 cells were analyzed using Western blotting. ( C , D ) mRNA expression of SREBP-1 and ACOX1 in the liver tissue of ND- and HFD-fed mice were obtained from the GSE32095 dataset (ND, n = 3; HFD, n = 3). Asterisks (*) indicate significant differences (*** p < 0.001) compared with the normal diet group. ( E ) mRNA expression of SIRT1-linked hepatic lipogenesis and fatty acid oxidation genes in the liver of control and SIRT1 knockout mice were obtained from the GSE14921 dataset (Control, n = 3; SIRT1 ko, n = 3). Asterisks (*) indicate significant differences (* p < 0.05 and ** p < 0.01) compared with the control group. ( F ) Protein expression levels of, ACOX1, CPT1α, and nuclear expression levels of SIRT1 in palmitate (500 µM)-treated HepG2 cells were analyzed using Western blotting. TFIIB was used as a loading control for the nuclear lysates. 8PG, 8-prenylgenistein; ACC, acetyl-CoA carboxylase; ACOX1, acyl-CoA oxidase-1; AMPK, adenosine 5′ monophosphate-activated protein kinase; CPT1α, carnitine palmitoyltransferase I alpha; FAS, fatty Acid Synthase; HFD, high-fat diet, ND, normal diet, SD, standard deviation; SIRT1, sirtuin 1; SREBP-1, sterol regulatory element-binding protein 1, TCGA, The Cancer Genome Atlas Program, TFIIB, transcription factor II b.

Article Snippet: Primary antibodies against the following proteins were used for Western blot analysis: AMPK, p-AMPK (Thr172), ACC, p-ACC (Ser79), CPT1α, SIRT1, TFIIB, and β-actin; these antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Activation Assay, Expressing, Standard Deviation, Western Blot, Control, Knock-Out, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: 8-Prenylgenistein Isoflavone in Cheonggukjang Acts as a Novel AMPK Activator Attenuating Hepatic Steatosis by Enhancing the SIRT1-Mediated Pathway

doi: 10.3390/ijms25179730

Figure Lengend Snippet: Schematic outlining the mechanism by which 8PG from CGJ mitigates hepatic steatosis. 8PG induced the activation of AMPK and its downstream target, ACC, and further increased the protein level of nuclear SIRT1, which is associated with the inhibition of hepatic lipogenesis and activation of fatty acid β-oxidation (CPT1α and ACOX1) in HepG2 cells, thereby counteracting hepatic steatosis. (↑ and ↓ indicate upregulation and downregulation, respectively). 8PG, 8-prenylgenistein; ACC, acetyl-CoA carboxylase; ACOX1, acyl-CoA oxidase-1; AMPK, adenosine 5′ monophosphate-activated protein kinase; CPT1α, carnitine palmitoyltransferase I alpha; FAS, fatty acid synthase; MASLD, metabolic dysfunction-associated steatotic liver disease; SIRT1, sirtuin 1; SREBP-1, sterol regulatory element-binding protein 1.

Article Snippet: Primary antibodies against the following proteins were used for Western blot analysis: AMPK, p-AMPK (Thr172), ACC, p-ACC (Ser79), CPT1α, SIRT1, TFIIB, and β-actin; these antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Activation Assay, Inhibition, Binding Assay