chrebp nb400-135 antibody  (Novus Biologicals)

Journal: The Journal of Biological Chemistry

Article Title: The sphingosine-1-phosphate receptor 2 S1PR2 mediates chronic glucocorticoid exposure-induced hepatic steatosis and hypertriglyceridemia

doi: 10.1016/j.jbc.2025.110353

Figure Lengend Snippet: RNA sequencing identifies ChREBP target genes to be activated by Dex and reduced by hepatic S1PR2 knockdown . A , volcano plot of differentially expressed genes in livers of shScr mice treated with or without Dex with 1.5-fold difference and adjusted p -value of 0.05 as a cutoff. B , gene ontology analysis by Kegg Pathways of genes induced or reduced between shScr mice treated with or without Dex. C , volcano plot of differentially expressed genes in livers of shScr and shS1PR2 Dex-treated mice with 1.5-fold difference and adjusted p -value of 0.05 as a cutoff. D , gene ontology analysis by Kegg Pathways of genes induced and reduced between livers of shScr and shS1PR2 Dex-treated mice. E , ChREBP target differentially expressed genes involved in glycolysis and fructolysis that are regulated by chronic Dex treatment and hepatic S1PR2 knockdown. Genes in red font are upregulated by chronic Dex treatment. Purple arrows and underlining indicate genes that are downregulated by hepatic S1PR2 knockdown. Figure created by BioRender. F , mRNA levels in shScr and shS1PR2 livers treated with or without Dex, n = 8 to 16. G , Relative lactate levels in livers of shScr and shS1PR2 mice treated with or without Dex, n = 8 to 16. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001 by two-way ANOVA with Fisher’s LSD test. Data presented as mean with S.D.

Article Snippet: Primary antibodies used are as follows: Alpha Tubulin (11224-1-AP, Proteintech, Rosemont, IL), S1PR2 (21180-1-AP, Proteintech), SPHK2 (ABS526, Millipore Sigma), ChREBP (NB400-135, Novus Biologicals).

Techniques: RNA Sequencing, Knockdown

Journal: The Journal of Biological Chemistry

Article Title: The sphingosine-1-phosphate receptor 2 S1PR2 mediates chronic glucocorticoid exposure-induced hepatic steatosis and hypertriglyceridemia

doi: 10.1016/j.jbc.2025.110353

Figure Lengend Snippet: Dex-induced ChREBP recruitment to target genes is reduced by hepatic S1PR2 knockdown. A , ChIP of ChREBP to ChoREs in livers of shScr and shS1PR2 mice treated with or without Dex, n = 7 to 10. B , hepatic mRNA levels in shScr and shS1PR2 mice treated with or without Dex, n = 8 to 19. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by two-way ANOVA with Fisher’s LSD test. C , representative western blots in liver of Dex-treated shScr and shS1PR2 mice and band density quantification by ImageJ, n = 9. D , mRNA levels in mouse primary hepatocytes treated with or without CYM-5520, n = 6. ∗ p < 0.05 by unpaired t test with Welch’s correction. Data presented as mean with S.D.

Article Snippet: Primary antibodies used are as follows: Alpha Tubulin (11224-1-AP, Proteintech, Rosemont, IL), S1PR2 (21180-1-AP, Proteintech), SPHK2 (ABS526, Millipore Sigma), ChREBP (NB400-135, Novus Biologicals).

Techniques: Knockdown, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The sphingosine-1-phosphate receptor 2 S1PR2 mediates chronic glucocorticoid exposure-induced hepatic steatosis and hypertriglyceridemia

doi: 10.1016/j.jbc.2025.110353

Figure Lengend Snippet: Model of chronic Dex exposure-induced S1PR2 signaling on hepatic steatosis and hypertriglyceridemia. Chronic Dex exposure induces S1PR2 signaling which activates ChREBP and Srebp1c to bind to their target promoter genes. These genes are involved in glycolysis and lipogenesis that contribute toward the lipid disorders of hepatic steatosis and hypertriglyceridemia. Figure created using BioRender.

Article Snippet: Primary antibodies used are as follows: Alpha Tubulin (11224-1-AP, Proteintech, Rosemont, IL), S1PR2 (21180-1-AP, Proteintech), SPHK2 (ABS526, Millipore Sigma), ChREBP (NB400-135, Novus Biologicals).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Hepatic ChREBP reciprocally modulates systemic insulin sensitivity in NAFLD

doi: 10.1016/j.jbc.2025.108556

Figure Lengend Snippet: Hepatic ChREBP deficiency improves insulin sensitivity in DIO mice directly by reducing PTEN expression. A , immunoblot images and quantification of ChREBP and PTEN from the liver lysates of all the mice groups discussed in A , quantified against GAPDH as the loading control (GAPDH used in K is reused for this blot because the data is from same blot and has the same protein quantification). B , immunoblot images and quantification of ChREBP and PTEN, quantified against GAPDH, from liver lysates of mice fed with RCD and 60% high fat diet only (HFD) for 6 weeks. C , immunoblot images and quantification of ChREBP and PTEN, quantified against GAPDH, from liver lysates of ob/ob mice against control C57BL/6 mice fed with RCD. D , schematic portrayal of PTEN promoter cloned in pGL3 vector having ChORE sequence and the ChORE deletion mutant. E , plot of the luciferase assay done in HepG2 cells at low (5 mM) and high (30 mM) glucose concentration after cloning the luciferase construct as shown in . F , ChIP analysis done in HepG2 cells for PTEN promoter occupancy upon low and high glucose concentration after pulling down with ChREBP antibody. G , plot of the luciferase assay at low (5 mM) and high (30 mM) glucose concentration, upon transfection with the WT and MUT into HepG2 cells. H , immunoblot image and quantification of PTEN upon ChREBP knockdown in HepG2 cells, quantified with GAPDH as the loading control. I , immunoblot image of PTEN upon ChREBP overexpression and knockdown in low and high glucose concentration in HepG2 cells. J , immunoblot images and quantification of ChREBP and PTEN in fasting (24 h) and refeeding (24 h) mice liver lysates from mice feeding on RCD and HFSD. K , immunoblot images and quantification of ChREBP and PTEN in fasting (24 h) and refeeding (24 h) mice liver lysates of mice given scramble shRNA (HFSD+shScr) versus mice with HFSD+shChREBP. D–I , are from in vitro HepG2 cells, and all other are from in vivo C57BL6 mice models. Mean ± SD. ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001.

Article Snippet: Chromatin immunoprecipitation was done using the ChREBP antibody (Abclonal, A7630) and q-PCR primers for the ChoRE-binding site on the PTEN promoter (FP-TTTCCGAGGCGCCCTGCT; RP-ATGGCTGCAGCTTCCGA).

Techniques: Expressing, Western Blot, Control, Clone Assay, Plasmid Preparation, Sequencing, Mutagenesis, Luciferase, Concentration Assay, Cloning, Construct, Transfection, Knockdown, Over Expression, shRNA, In Vitro, In Vivo

Journal: Journal of molecular biology

Article Title: An acetylated lysine residue of its low-glucose inhibitory domain controls activity and protein interactions of ChREBP.

doi: 10.1016/j.jmb.2025.169189

Figure Lengend Snippet: Figure 2. Mutation of K171 enhances ChREBP activity. (A) HEK293 cells were transfected with vectors encoding wt or mutated ChREBP and exposed to 2.5 mM or 25 mM of glucose for 24 hours and mRNA expression determined by qPCR. (B) ChREBP protein expression of cells described in (A) was analyzed by immunoblottiong. ACTB served as loading control. (C) Cells described in (A) were cotransfected with a ChoRE-driven luciferase reporter and analyzed for its activity. (D) Cells described in (A) were analyzed for mRNA expression of hTXNIP by qPCR. (E) HEK293 cells were transfected with vectors encoding wt or mutated ChREBP and its protein expression analyzed by immunoblottiong. ACTB served as loading control. (F) Cells described in (E) were cotransfected with a carbohydrate

Article Snippet: Another limitation is in regard to the dynamics of K171 acetylation, which we could not assess due to the lack of a specific antibody for acetylated K171 of ChREBP and poor, unreproducible results using a pan-acetylated-lysine antibody (Cell Signaling #9681, lot.

Techniques: Mutagenesis, Activity Assay, Transfection, Expressing, Control, Luciferase

Journal: Journal of molecular biology

Article Title: An acetylated lysine residue of its low-glucose inhibitory domain controls activity and protein interactions of ChREBP.

doi: 10.1016/j.jmb.2025.169189

Figure Lengend Snippet: Figure 3. Mutation of K171 increases nuclear localization and genomic binding of ChREBP. (A) HEK293 cells were transfected with vectors encoding wt or mutated ChREBP and exposed to 2.5 mM or 25 mM of glucose for 24 hours. Subsequently, subcellular fractions were isolated and cytosolic and nuclear proteins analyzed for ChREBP abundance by immunoblottinging. GAPDH and Histone H3 served as loading and fractionation controls. (B) Densitometric analysis of total ChREBP protein levels in cells described in (A) of 3 independent experiments. (C) Densitometric analysis of cytosolic and nuclear ChREBP protein abundance in cells described in (A) of 3 independent experiments. (D) Cells described in (A) were analyzed for ChREBP binding to a carbohydrate response element (ChoRE) upstream of the transcriptional start site of the hTXNIP gene by chromatin immunoprecipitation (ChIP). Data are represented as individual data points and mean ± sem and *P<0.05 vs. wt ChREBP-expressing cells by one-way ANOVA and Sidak’s correction for multiple testing.

Article Snippet: Another limitation is in regard to the dynamics of K171 acetylation, which we could not assess due to the lack of a specific antibody for acetylated K171 of ChREBP and poor, unreproducible results using a pan-acetylated-lysine antibody (Cell Signaling #9681, lot.

Techniques: Mutagenesis, Binding Assay, Transfection, Isolation, Fractionation, Quantitative Proteomics, Chromatin Immunoprecipitation, Expressing

Journal: Journal of molecular biology

Article Title: An acetylated lysine residue of its low-glucose inhibitory domain controls activity and protein interactions of ChREBP.

doi: 10.1016/j.jmb.2025.169189

Figure Lengend Snippet: Figure 4. Identifying the ChREBP protein interactome and its differential binding in HEK293 cells. HEK293 cells were transfected with vectors encoding Flag-tagged wt or mutated ChREBP and exposed to 2.5 mM or 25 mM of glucose for 24 hours. Flag-immunoprecipitated (IP) proteins were analyzed by mass spectrometry. Flag-IP protein of empty vector-transfected cells served as negative control. (A) Venn diagram of total number of identified interacting proteins (FDR 5%). (B) Analysis of the Top 5-enriched pathways (top panel), protein domains (middle panel), and regulating transcription factors (bottom panel) of n=333 proteins that interact with both wt and K171R ChREBP. Clustered heatmaps of (C) n=83 proteins that show significant differential binding (FDR 10%) between wt and K171 ChREBP and (E) of n=26 proteins that show significant differential binding between 2.5 mM and 25 mM glucose (FDR 10%) of wt (n=17) or K171R ChREBP (n=9). Top 10-simplified GO: Cellular Component terms of (D) ChREBP wt vs. K171R interactors and of (F) glucose differential interactors at 2.5 mM glucose. Differential protein abundance was calculated using student´s t test with a cut off for interactor definition against control of FDR 5% and of FDR 10% for differential interactors between conditions. (D) and (F) depict FDR 5% TOP10 GO terms.

Article Snippet: Another limitation is in regard to the dynamics of K171 acetylation, which we could not assess due to the lack of a specific antibody for acetylated K171 of ChREBP and poor, unreproducible results using a pan-acetylated-lysine antibody (Cell Signaling #9681, lot.

Techniques: Binding Assay, Transfection, Immunoprecipitation, Mass Spectrometry, Plasmid Preparation, Negative Control, Quantitative Proteomics, Control

Journal: Journal of molecular biology

Article Title: An acetylated lysine residue of its low-glucose inhibitory domain controls activity and protein interactions of ChREBP.

doi: 10.1016/j.jmb.2025.169189

Figure Lengend Snippet: Figure 5. Mutation of ChREBP K171 reduces physical interaction with 14-3-3 proteins and affects interaction with proteins involved in nuclear import and the NuRD complex. HEK293 cells were transfected with vectors encoding Flag-tagged wt or mutated ChREBP and exposed to 2.5 mM or 25 mM of glucose for 24 hours. (A) Flag-IP'ed proteins were analyzed by mass spectrometry (MassSpec) for interaction with 14-3-3 isoforms. (B) Flag-immunoprecipiated (IP) proteins were analyzed for ChREBP and 14-3-3 protein by immunoblotting. Flag-IP protein of empty vector-transfected cells served as negative control. GAPDH served as loading and IP control. (C) Densitometric analysis of co- IP'ed 14-3-3 protein shown in (B) of 4 independent experiments. (D-F) Clustered heatmaps of ChREBP- interacting proteins based on GO terms that relate to nuclear translocation (D, E) and the NuRD complex (F). In (C), data are presented as individual data points and mean ± sem and with *P<0.05 vs. wt ChREBP-expressing cells by one-way ANOVA and Sidak’s correction for multiple testing. Differential protein abundance was calculated using student´s t test with a cut off for interactor definition against control of FDR 5% and of FDR 10% for differential interactors between wt and K171R within the GO term.

Article Snippet: Another limitation is in regard to the dynamics of K171 acetylation, which we could not assess due to the lack of a specific antibody for acetylated K171 of ChREBP and poor, unreproducible results using a pan-acetylated-lysine antibody (Cell Signaling #9681, lot.

Techniques: Mutagenesis, Transfection, Mass Spectrometry, Western Blot, Plasmid Preparation, Negative Control, Control, Co-Immunoprecipitation Assay, Translocation Assay, Expressing, Quantitative Proteomics