Journal: Molecular and Cellular Biology

Article Title: Retinol-Binding Protein 4 Induces Inflammation in Human Endothelial Cells by an NADPH Oxidase- and Nuclear Factor Kappa B-Dependent and Retinol-Independent Mechanism

doi: 10.1128/MCB.00820-12

Figure Lengend Snippet: RBP4 induces expression of proinflammatory cell adhesion molecules and monocyte chemoattractant protein-1 in human endothelial cells in a dose-dependent manner. (A) Representative Coomassie blue staining and Western blot analysis of purified recombinant human holo-RBP4 (1 μg per lane). Lane 1, Coomassie blue-stained gel; lane 2, Western blot with RBP4 antibody. (B) UV spectrum analysis of purified recombinant holo-RBP4 demonstrates a retinol/RBP4 ratio of approximately 0.9, indicating that the majority of the purified RBP4 consists of holo-RBP4. (C to F) Quantitative RT-PCR analysis for mRNA expression of VCAM-1, ICAM-1, E-selectin, and MCP-1 in human retinal capillary endothelial cells (HRCEC) treated with increasing concentrations of holo-RBP4 (10 to 100 μg/ml) or BSA or left untreated (Unt.). Significant differences compared to BSA treatment: *, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way analysis of variance (ANOVA) with Tukey's post hoc test.

Article Snippet: Native human RBP4, which was purified from normal human serum, was obtained from Athens Research Technology (Athens, GA).

Techniques: Expressing, Staining, Western Blot, Purification, Recombinant, Quantitative RT-PCR

Journal: Molecular and Cellular Biology

Article Title: Retinol-Binding Protein 4 Induces Inflammation in Human Endothelial Cells by an NADPH Oxidase- and Nuclear Factor Kappa B-Dependent and Retinol-Independent Mechanism

doi: 10.1128/MCB.00820-12

Figure Lengend Snippet: RBP4 activates NADPH oxidase protein expression and stimulates proinflammatory protein expression via an NADPH oxidase-dependent mechanism in HRCEC. (A and B) HRCEC were treated with BSA or increasing concentrations of holo-RBP4 for 24 h, and cell lysates were analyzed by Western blotting for Nox2 (A) and Nox4 (B). (C through H) HRCEC were pretreated with NADPH oxidase inhibitors DPI (20 μM) or apocynin (500 or 1,000 μM) for 2 h prior to the addition of holo-RBP4 at 100 μg/ml for 24 h. (C and D) Western blots of VCAM-1 following treatment with RBP4 in the absence or presence of DPI (C) or apocynin (D). (E through H) ELISA-based quantification of soluble extracellular levels of sVCAM-1 (E), sICAM-1 (F), MCP-1 (G), and E-selectin (H) in HRCEC media. Significant differences compared to RBP4 treatment alone: *, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way ANOVA with Tukey's post hoc test.

Article Snippet: Native human RBP4, which was purified from normal human serum, was obtained from Athens Research Technology (Athens, GA).

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Molecular and Cellular Biology

Article Title: Retinol-Binding Protein 4 Induces Inflammation in Human Endothelial Cells by an NADPH Oxidase- and Nuclear Factor Kappa B-Dependent and Retinol-Independent Mechanism

doi: 10.1128/MCB.00820-12

Figure Lengend Snippet: RBP4 increases cellular and extracellular protein levels of proinflammatory molecules in HRCEC in a dose- and time-dependent manner. (A to C) Western blots of VCAM-1 (A), E-selectin (B), and ICAM-1(C) from HRCEC treated with BSA or increasing concentrations of RBP4 for 24 h. (D to F) Western blots of VCAM-1 (D), E-selectin (E), and ICAM-1 (F) from HRCEC treated with RBP4 at 100 μg/ml for the indicated times of 0 to 48 h. (G to J) ELISA-based quantification of soluble extracellular levels of sVCAM-1 (G), E-selectin (H), sICAM-1 (I), and MCP-1 (J) from HRCEC media following treatment with increasing concentrations of holo-RBP4 or BSA or no treatment (Unt.). (K to N) ELISA-based quantification of soluble extracellular levels of sVCAM-1 (K), E-selectin (L), sICAM-1 (M), and MCP-1 (N) in HRCEC media following treatment with holo-RBP4 at 100 μg/ml for the indicated times of 0 to 48 h. Significant differences compared to BSA treatment: *, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way ANOVA with Tukey's post hoc test.

Article Snippet: Native human RBP4, which was purified from normal human serum, was obtained from Athens Research Technology (Athens, GA).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Molecular and Cellular Biology

Article Title: Retinol-Binding Protein 4 Induces Inflammation in Human Endothelial Cells by an NADPH Oxidase- and Nuclear Factor Kappa B-Dependent and Retinol-Independent Mechanism

doi: 10.1128/MCB.00820-12

Figure Lengend Snippet: RBP4 increases leukocyte adherence to human endothelial cells. Confluent monolayers of HRCEC were treated with either holo-RBP4 (100 μg/ml), BSA, or TNF-α (100 ng/ml) for 18 h. THP-1 monocytes were then added and cocultured for 3 h. (A) Representative phase-contrast images (magnification, ×20) of monocyte adherence to HRCEC after the indicated treatment. Adherent monocytes are apparent as small bright circular bodies above the HRCEC layer. (B) Adherent monocytes were counted per visual field at magnification of ×20. The graph shows the means ± standard deviations from 4 different visual fields for each treatment group (adherent leukocytes in TNF-α positive control were 205 ± 25 per visual field). ***, P < 0.001 versus BSA treatment by one-way ANOVA with Tukey's post hoc test. Untreat, untreated cells.

Article Snippet: Native human RBP4, which was purified from normal human serum, was obtained from Athens Research Technology (Athens, GA).

Techniques: Positive Control

Journal: Molecular and Cellular Biology

Article Title: Retinol-Binding Protein 4 Induces Inflammation in Human Endothelial Cells by an NADPH Oxidase- and Nuclear Factor Kappa B-Dependent and Retinol-Independent Mechanism

doi: 10.1128/MCB.00820-12

Figure Lengend Snippet: RBP4 activates NF-κB and stimulates proinflammatory protein expression at least partially through an NF-κB-dependent mechanism in HRCEC. (A) Nuclear translocation of NF-κB p65 detected by immunocytochemistry in HRCEC after treatment with holo-RBP4 (100 μg/ml), BSA, or TNF-α for 12 h; untreat, untreated cells. (B) The relative nuclear transcriptional activity of NF-κB p65 was quantified using the ELISA-based TransAM NF-κB assay. HRCEC were treated with BSA or holo-RBP4 (100 μg/ml) for 6 h, and nuclear extracts were prepared and analyzed. Western blots of beta-actin, GAPDH, and fibrillarin demonstrate the enrichment and quality of nuclear extract preparations. Lane C, cytosolic extract; lane N, nuclear extract (10 μg protein/lane). (C) HRCEC were treated with either BSA or holo-RBP4 (100 μg/ml) for 24 h, and phosphorylation of NF-κB p65 at Ser536 was quantified by Western blotting and band densitometry analysis. (D through I) HRCEC were pretreated with NF-κB inhibitors PDTC (100 or 500 μM) or JSH-23 (10 or 50 μM) for 2 h prior to the addition of holo-RBP4 at 100 μg/ml for 24 h. (D through E) Western blots of VCAM-1 following treatment with RBP4 in the absence or presence of PDTC (D) or JSH-23 (E). (F through I) ELISA-based quantification of soluble extracellular levels of sVCAM-1 (F), sICAM-1 (G), MCP-1 (H), and E-selectin (I) in HRCEC media. Significant differences compared to RBP4 treatment alone: *, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way ANOVA with Tukey's post hoc test. Significant difference compared to BSA treatment (panels B and C): †, P < 0.01 by Student's t test.

Article Snippet: Native human RBP4, which was purified from normal human serum, was obtained from Athens Research Technology (Athens, GA).

Techniques: Expressing, Translocation Assay, Immunocytochemistry, Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Phospho-proteomics

Journal: Molecular and Cellular Biology

Article Title: Retinol-Binding Protein 4 Induces Inflammation in Human Endothelial Cells by an NADPH Oxidase- and Nuclear Factor Kappa B-Dependent and Retinol-Independent Mechanism

doi: 10.1128/MCB.00820-12

Figure Lengend Snippet: RBP4-mediated activation of NF-κB, NADPH oxidase, and proinflammatory molecules is retinol independent. (A) Quantitative RT-PCR analysis of STRA6 mRNA expression in HRCEC following 24 h of treatment with RBP4 (100 μg/ml) or BSA or no treatment (Untr.). STRA6 mRNA expression is shown relative to HPRT housekeeping gene expression and is 20-fold less abundant than HPRT. (B) STRA6 protein expression was undetectable by Western blotting of HRCEC. HEK-293A cells stably expressing STRA6 were also analyzed as a positive control. (C) HRCEC intracellular retinoid content following treatment with holo-RBP4 (100 μg/ml) or an equimolar amount (4.75 μM) of retinol, retinal, or retinoic acid. HRCEC did not uptake a significant amount of retinol from holo-RBP4. (D and E) Western blots of VCAM-1 and Nox2 (D) or Nox4 (E) in HRCEC treated with increasing concentrations of apo-RBP4 for 24 h. (F) HRCEC were treated with either BSA or apo-RBP4 (100 μg/ml) for 24 h, and phosphorylation of NF-κB p65 at Ser536 was quantified by Western blotting and band densitometry analysis. (G through J) ELISA-based quantification of soluble extracellular levels of MCP-1 (G), sVCAM-1 (H), sICAM-1 (I), and E-selectin (J) in HRCEC media following 24 h of treatment with increasing concentrations of either apo- or holo-RBP4 as indicated. Significant differences compared to BSA treatment: *, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way ANOVA with Tukey's post hoc test.

Article Snippet: Native human RBP4, which was purified from normal human serum, was obtained from Athens Research Technology (Athens, GA).

Techniques: Activation Assay, Quantitative RT-PCR, Expressing, Gene Expression, Western Blot, Stable Transfection, Positive Control, Phospho-proteomics, Enzyme-linked Immunosorbent Assay

Journal: Molecular and Cellular Biology

Article Title: Retinol-Binding Protein 4 Induces Inflammation in Human Endothelial Cells by an NADPH Oxidase- and Nuclear Factor Kappa B-Dependent and Retinol-Independent Mechanism

doi: 10.1128/MCB.00820-12

Figure Lengend Snippet: RBP4 induces NF-κB, NADPH oxidase, and proinflammatory molecules in human umbilical vein endothelial cells. (A through C) ELISA-based quantification of soluble extracellular levels of sVCAM-1 (A), sICAM-1 (B), and MCP-1 (C) in HUVEC media following 24 h of treatment with increasing concentrations of either apo- or holo-RBP4 as indicated. (D) Western blots of E-selectin and Nox2, VCAM-1, and Nox4 in HUVEC treated with increasing concentrations of holo-RBP4 for 24 h. (E) Nuclear translocation of NF-κB p65 detected by immunocytochemistry in HUVEC after treatment with holo-RBP4 (100 μg/ml), BSA, or TNF-α for 12 h. Untreat, untreated cells. (F) HUVEC were treated with either BSA or apo-RBP4 (100 μg/ml) for 24 h, and phosphorylation of NF-κB p65 at Ser536 was quantified by Western blotting and band densitometry analysis. Significant differences compared to BSA treatment: *, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way ANOVA with Tukey's post hoc test; †, P < 0.01 by Student's t test.

Article Snippet: Native human RBP4, which was purified from normal human serum, was obtained from Athens Research Technology (Athens, GA).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Translocation Assay, Immunocytochemistry, Phospho-proteomics

Journal: Molecular and Cellular Biology

Article Title: Retinol-Binding Protein 4 Induces Inflammation in Human Endothelial Cells by an NADPH Oxidase- and Nuclear Factor Kappa B-Dependent and Retinol-Independent Mechanism

doi: 10.1128/MCB.00820-12

Figure Lengend Snippet: RBP4-mediated induction of proinflammatory proteins in HUVEC is via activation of NADPH oxidase and NF-κB. (A through C) HUVEC were pretreated with NF-κB inhibitors PDTC (100 or 500 μM) or JSH-23 (10 or 50 μM) or with NADPH oxidase inhibitors DPI (20 μM) or apocynin (500 or 1,000 μM) for 2 h prior to the addition of holo-RBP4 at 100 μg/ml for 24 h. ELISA-based quantification of soluble extracellular levels of sICAM-1 (A), sVCAM-1 (B), and MCP-1 (C) in HUVEC media. Significant differences compared to RBP4 treatment alone: *, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way ANOVA; †, P < 0.01 by Student's t test. (D and E) Western blots of VCAM-1 in HUVEC following RBP4 treatment in the presence or absence of PDTC (D) or DPI (E).

Article Snippet: Native human RBP4, which was purified from normal human serum, was obtained from Athens Research Technology (Athens, GA).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Circulation research

Article Title: RBPMS and RBPMS2 Cooperate to Safeguard Cardiac Splicing

doi: 10.1161/CIRCRESAHA.125.326948

Figure Lengend Snippet: (A) RBPMS and RBPMS2 (green) are localized to the nuclei (DAPI, grey) of cardiomyocytes (CMs, marked by Nkx2–5, red). (B) RBPMS and RBPMS2 (green) are localized to the nuclei of vascular smooth muscle cells (SMCs, marked by αSMA/α smooth muscle actin, red) in coronary vessels and aorta. (C) RBPMS and RBPMS2 (green) are localized to the nuclei of endothelial cells (ECs, marked by endomucin/EMCN, blue). (D) RBPMS and RBPMS2 (green) are localized to the nuclei of fibroblasts (FBs, marked by PDGFRα, red). Yellow arrows: CM nuclei; Orange arrows: SMC nuclei; Blue arrows: EC nuclei; Magenta arrows: FB nuclei. Rbpms global KO samples ( Rbpms gKO ) were used as negative controls. Scale bar: 20 μm.

Article Snippet: Sources for antibodies used in immunofluorescence in this study: pan-RBPMS, 15187–1-AP (Proteintech); Nkx2–5, sc-8697 (Santa Cruz Biotechnology); EMCN, 14–5851-82 (Invitrogen); αSMA, ab7817 (Abcam); PDGFRα, AF1062 (R&D System); α-actinin-2, ab68167 (Abcam); Myom1, B4 (DSHB).

Techniques: Expressing

Journal: Circulation research

Article Title: RBPMS and RBPMS2 Cooperate to Safeguard Cardiac Splicing

doi: 10.1161/CIRCRESAHA.125.326948

Figure Lengend Snippet: (A) Kaplan-Meier survival curves of control (n=7) and Rbpms cmKO (n=5) mice. (B) Representative hematoxylin and eosin (H&E)-stained sections from control and Rbpms cmKO mice at embryonic day 17.5 (E17.5) and postnatal day 30 (P30). Orange rulers indicate left ventricle (LV) trabeculae thickness while red rulers indicate LV compact layer thickness; Scale bar: 1 mm (whole heart view); 0.5 mm (magnified view). (C) Quantification of the thickness of the LV compact layer (LV-Com), LV trabeculae (LV-Tra), and interventricular septum (IVS) on H&E sections from control (n=5) and Rbpms cmKO (n=4) mice at E17.5. p-values were determined by Mann-Whitney test. (D) Echocardiography analysis of control (n=7) and Rbpms cmKO (n=5) mice at P30. p-values were determined by Mann-Whitney test. FS: fraction shortening; LV-IDd/s: LV internal dimension, diastolic/systolic; LV-PWd: LV posterior wall thickness, diastolic; IVSd: interventricular septum thickness, diastolic. (E) Left panel: representative immunostaining images of LV-com of E17.5 control and Rbpms cmKO mice using antibodies against Nkx2–5 (red), EdU signal (green), and DAPI (blue). Arrows indicate EdU-positive cardiomyocytes (CMs). Scale bar: 50 μm. Right panel: quantification (%) of EdU-positive CMs in E17.5 control (n=4) vs. Rbpms cmKO (n=4) heart areas as indicated. p-values were determined by Mann-Whitney test.

Article Snippet: Sources for antibodies used in immunofluorescence in this study: pan-RBPMS, 15187–1-AP (Proteintech); Nkx2–5, sc-8697 (Santa Cruz Biotechnology); EMCN, 14–5851-82 (Invitrogen); αSMA, ab7817 (Abcam); PDGFRα, AF1062 (R&D System); α-actinin-2, ab68167 (Abcam); Myom1, B4 (DSHB).

Techniques: Control, Staining, MANN-WHITNEY, Immunostaining

Journal: Cell Death & Disease

Article Title: The RBPJ/DAPK3/UBE3A signaling axis induces PBRM1 degradation to modulate the sensitivity of renal cell carcinoma to CDK4/6 inhibitors

doi: 10.1038/s41419-022-04760-6

Figure Lengend Snippet: a Bioinformatic analysis of the potential binding proteins of the promoter of DAPK3 via analysis the ChIP-seq datasets. b , c 786-O and ACHN cells were transfected with indicated plasmids for 24 h. Cells were harvested for western blotting analysis ( b ) and RT-qPCR analysis ( c ). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SEM with three replicates ( n = 3). Ns not significant; ** P < 0.01; *** P < 0.001. d , e 786-O and ACHN cells were infected with indicated shRNAs for 72 h. Cells were harvested for western blotting analysis ( d ) and RT-qPCR analysis ( e ). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SEM with three replicates ( n = 3). ** P < 0.01. f , g 786-O cells were treated with 0, 1 µM, 5 µM, and 10 µM the RBPJ inhibitors (the DAPK3 inhibitor) for 24 h. The WCL of cells were subjected to western blotting analysis ( f ) and RT-qPCR analysis ( g ). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SEM with three replicates ( n = 3). Ns not significant; *** P < 0.001. h the ChIP-seq of RBPJ on the promoter region of DAPK3 . i 786-O cells were infected with indicated shRNAs for 72 h. Cells were harvested for ChIP-qPCR analysis by using the IgG or RBPJ antibodies. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SEM with three replicates ( n = 3). ** P < 0.01; *** P < 0.001. j 786-O cells were transfected with indicated plasmids for 24 h. Cells were harvested for ChIP-qPCR analysis by using the IgG or RBPJ antibodies. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SEM with three replicates ( n = 3). * P < 0.05; ** P < 0.01; *** P < 0.001. k The correlation between RBPJ and DAPK3 were analyzed by the GEPIA web tool ( http://gepia.cancer-pku.cn/ ) in different types of cancer. l 786-O cells were infected with indicated shRNAs for 72 h. Cells were harvested for western blotting analysis. m 786-O cells were infected with indicated shRNAs for 48 h. Then, cells were treated with or without the 5 µM RBPJ inhibitor for another 24 h. Cells were harvested for western blotting analysis.

Article Snippet: The antibodies used as follows: UBE3A (10344-1-AP, Proteintech; 1:1000 dilution), PBRM1 (12563-1-AP, Proteintech; 1:500 dilution), DAPK3 (2928, Cell signaling technology, 1:1000 dilution), RBPJ (14613-1-AP, Proteintech; 1:1000 dilution); P21 (10355-1-AP, Proteintech; 1:1000 dilution); GAPDH (10494-1-AP, Proteintech; 1:10000 dilution).

Techniques: Binding Assay, ChIP-sequencing, Transfection, Western Blot, Quantitative RT-PCR, Infection, ChIP-qPCR

Journal: Cell Death & Disease

Article Title: The RBPJ/DAPK3/UBE3A signaling axis induces PBRM1 degradation to modulate the sensitivity of renal cell carcinoma to CDK4/6 inhibitors

doi: 10.1038/s41419-022-04760-6

Figure Lengend Snippet: DAPK3 competed with PKA to bind with UBE3A and enhance the PBRM1 degradation in renal cancer cells. PBPJ transcriptionally regulated DAPK3 expression and then promoted UBE3A-mediated degradation of PBRM1. Then, PBRM1 increased the p21 expression and sensitized renal cancer cells to CDK4/6 inhibitors. In combination with RBPJ inhibitors, CDK4/6 inhibitors synergistically enhanced renal cancer cells.

Article Snippet: The antibodies used as follows: UBE3A (10344-1-AP, Proteintech; 1:1000 dilution), PBRM1 (12563-1-AP, Proteintech; 1:500 dilution), DAPK3 (2928, Cell signaling technology, 1:1000 dilution), RBPJ (14613-1-AP, Proteintech; 1:1000 dilution); P21 (10355-1-AP, Proteintech; 1:1000 dilution); GAPDH (10494-1-AP, Proteintech; 1:10000 dilution).

Techniques: Expressing

Journal: Annals of clinical and translational neurology

Article Title: Proteomics and mathematical modeling of longitudinal CSF differentiates fast versus slow ALS progression.

doi: 10.1002/acn3.51890

Figure Lengend Snippet: Figure 6. Receiver operator characteristic (ROC) curves using targeted measurements of candidate markers of progression by ELISA in the (A) discovery and (B) validation cohorts. Respective area under the curve (AUC) for SERPINA4 (green), RBP4 (red), F12 (blue), and combination (purple) are provided.

Article Snippet: Measurements of human retinol binding protein 4 (RBP4) and kallistatin (SERPINA4) were performed using DuoSet ELISA kits (R&D systems; Minneapolis,per MN) following the manufacturer’s protocol.

Techniques: Enzyme-linked Immunosorbent Assay, Biomarker Discovery

Journal: Nature Communications

Article Title: Alteration of circadian machinery in monocytes underlies chronic kidney disease-associated cardiac inflammation and fibrosis

doi: 10.1038/s41467-021-23050-x

Figure Lengend Snippet: a Functional analysis of the genes using the KEGG database. Detailed data are presented in Supplementary Table . b , c The mRNA levels of Gpr68 in mouse primary cultured monocytes after treatment with TNFα, IL-6, angiotensin II, or retinol/RBP4 at the indicated concentrations for 24 h. d – f The protein levels of GPR68 and phosphorylated STAT5 (phos-STAT5), and the mRNA levels of Clock and Arntl in mouse primary cultured monocytes after treatment with 1 or 3 µM of retinol/RBP4 for 24 h. g Schematic representation of the upstream region of the mouse Gpr68 , Clock , and Arntl genes. The numbers indicate the distance from the transcription start site (+1). The circled letters (orange circles) indicate the location on the gene where each of the different primer sets localize for analysis of ChIP. h , i ChIP analysis of retinol/RBP4-induced changes in the binding amount of STAT5 protein to the upstream region of Clock and Arntl genes in mouse primary cultured monocytes or the collected monocytes prepared from Sham and 5/6Nx mice. The primer sets used qPCR are illustrated in g . j ChIP analysis of retinol/RBP4-induced changes in the binding amount of CLOCK and ARNTL protein to the upstream region of Gpr68 genes in mouse primary cultured monocytes. Using primer sets illustrated in Fig. . k Left panel shows the protein levels of STRA6 in monocytes transfected with siRNA against STRA6 (uncropped images are presented in Supplementary Fig. ). Right panels show STRA6-dependent phosphorylation of STAT5 and expression of GPR68, CLOCK, and ARNTL by serum from Sham and 5/6Nx mice in mouse primary cultured monocytes. For all panels, graphs show the mean ± SD of individual mice in independent experiments ( h , j n = 5; the others n = 4 for each group). Statistical significance was determined using one-way ANOVA with Dunnett’s post hoc tests ( c ), two-tailed Student’s t -tests ( d – f , h – j ), or two-way ANOVA with Tukey–Kramer post hoc tests ( k ). Numbers and P -values are shown in the graph. Source data are provided as a Source Data file.

Article Snippet: The calibration curve for calculating RBP4 concentrations was made by the quantified values of recombinant RBP4 protein levels (mouse; R&D systems, Inc., Minneapolis, MN, USA.

Techniques: Functional Assay, Cell Culture, Binding Assay, Transfection, Phospho-proteomics, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Alteration of circadian machinery in monocytes underlies chronic kidney disease-associated cardiac inflammation and fibrosis

doi: 10.1038/s41467-021-23050-x

Figure Lengend Snippet: a Correlation between serum retinol and RBP4 levels in 5/6Nx mice fed with normal or vitamin A-free diet. b Dietary deficiency of vitamin A suppresses the upregulation of Gpr68 , Clock , and Arntl mRNA levels in circulating monocytes. The mean value of the Sham group was set as 1.0. c Double immunofluorescence labeling of GPR68 with F4/80 in the ventricle slices prepared from 5/6Nx mice fed with normal or Vitamin A-free diet. High-GPR68-expressing cells are double-labeled (yellow) with F4/80. The scale bar indicates 50 μm. d Serum BNP concentrations in Sham and 5/6Nx mice fed with normal or vitamin A-free diet. e Dietary deficiency of vitamin A ameliorates CKD-induced cardiac fibrosis. The Masson’s trichrome staining show tissue fibrosis in blue. Scale bars indicate 1 mm (upper panel) and 50 μm (lower panel). For all panels, graphs show the mean ± SD of individual mice in independent experiments. Statistical significance was determined using one-way ANOVA with Tukey–Kramer post hoc tests ( a , b , d ). Numbers and P -values are shown in each graph. Source data are provided as a Source Data file.

Article Snippet: The calibration curve for calculating RBP4 concentrations was made by the quantified values of recombinant RBP4 protein levels (mouse; R&D systems, Inc., Minneapolis, MN, USA.

Techniques: Immunofluorescence, Labeling, Expressing, Staining

Journal: Nature Communications

Article Title: Alteration of circadian machinery in monocytes underlies chronic kidney disease-associated cardiac inflammation and fibrosis

doi: 10.1038/s41467-021-23050-x

Figure Lengend Snippet: a Dose-dependent increase in the expression of GPR68 mRNA (left) and its protein (right) in human primary cultured monocytes by treatment with retinol/RBP4 for 24 h. b The phosphorylation state of STAT5 in human primary cultured monocytes after treatment with retinol/RBP4. c Schematic representation of the upstream region of the human GPR68 , CLOCK , and ARNTL genes. The circled letters (green circles) indicate the location on the gene where each of the different primer sets localize. d The binding of CLOCK, ARNTL, and STAT5 to regions, described in c , in human (healthy) primary cultured monocytes after treatment with retinol/RBP4 for 24 h. The primer sets used in qPCR are illustrated in c . e LPS-stimulated release of TNFα and IL-6 from human primary cultured monocytes after treatment with retinol/RBP4 for 24 h. f Serum retinol and RBP4 levels in CKD patients. The upper panel shows relationship between retinol and RBP4 levels. Lower panels show these levels stratified by serum creatinine levels (Scr). g , h Relationship retinol levels to mRNA expressions of GPR68 , IL-6 , TNFα , CLOCK , or ARNTL in human primary cultured monocytes incubated in media containing 20% serum collected from healthy subjects and CKD patients stratified by Scr. The release of TNFα and IL-6 from human primary cultured monocytes were assessed after incubation in the media containing 20% serum for 24 h. i Comparison of the values stratified by BNP concentrations. Left panel shows the expression levels of GPR68 mRNA in human primary cultured monocytes incubated same as in g . Right panel shows serum retinol levels. For panel f – i , each plot shows a value obtained using an individual human serum. For all panels, graphs show the mean ± SD in independent experiments ( a , b , d , e n = 5 for each group, f – i shown in each graph). Statistical significance was determined using one-way ANOVA with Dunnett’s post hoc tests ( a ), two-tailed Student’s t -tests ( a , b , d , g ), or one-way ANOVA with Tukey–Kramer post hoc tests ( e , i ). Numbers and P -values are shown in each graph.

Article Snippet: The calibration curve for calculating RBP4 concentrations was made by the quantified values of recombinant RBP4 protein levels (mouse; R&D systems, Inc., Minneapolis, MN, USA.

Techniques: Expressing, Cell Culture, Phospho-proteomics, Binding Assay, Incubation, Comparison, Two Tailed Test

Journal: Nature Communications

Article Title: Alteration of circadian machinery in monocytes underlies chronic kidney disease-associated cardiac inflammation and fibrosis

doi: 10.1038/s41467-021-23050-x

Figure Lengend Snippet: Serum levels of retinol and RBP4 were increased during chronic renal failure. The retinol-bound RBP4 activates JAK2/STAT5 signaling in circulating monocytes through STRA6 and induces the expression of GPR68 via activation of CLOCK/ARNTL. The high-GPR68-expressing monocytes infiltrate into the heart potential for producing inflammatory cytokines and their cardiac infiltration exacerbates inflammation and fibrosis.

Article Snippet: The calibration curve for calculating RBP4 concentrations was made by the quantified values of recombinant RBP4 protein levels (mouse; R&D systems, Inc., Minneapolis, MN, USA.

Techniques: Expressing, Activation Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Post transcriptional regulation of HLA-A protein expression by alternative polyadenylation signals involving the RNA binding protein Syncrip

doi: 10.4049/jimmunol.1700697

Figure Lengend Snippet: RNA was obtained from peripheral blood lymphocytes of healthy donors. Rapid amplification of cDNA ends (3’ RACE) of 3’UTRs of HLA-A alleles from 6 individuals with distinct HLA-A genotypes showed preferential use of either the proximal or distal PAS. HLA-A*01 and A*11 use only the proximal PAS, resulting in the short form of the 3’UTR, whereas others use various fractions of each. HLA-A genotypes of the individuals are indicated below each amplicon.

Article Snippet: EBV transformed B lymphoblastoid cell lines derived from individuals with known HLA-A genotypes ( HLA-A*11 or A*03 homozygotes) were transfected with a plasmid encoding Syncrip cDNA with a Myc-tag (RC217902, Origene) or a control plasmid (pcDNA3.1-eGFP, Addgene).

Techniques: Rapid Amplification of cDNA Ends, Amplification

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Post transcriptional regulation of HLA-A protein expression by alternative polyadenylation signals involving the RNA binding protein Syncrip

doi: 10.4049/jimmunol.1700697

Figure Lengend Snippet: (A) Western blot indicated the presence of Syncrip bound to the long form of HLA-A 3’UTR. RNA affinity pulldown was carried out with the in vitro transcribed 3’UTR fragments of canonical A*03 (PASPD) and PASDΔ300–325, labelled with biotin, and incubated with Jurkat cell lysate. RNA bound protein was separated on PAGE and western blot was carried out using anti-Syncrip antibody.

Article Snippet: EBV transformed B lymphoblastoid cell lines derived from individuals with known HLA-A genotypes ( HLA-A*11 or A*03 homozygotes) were transfected with a plasmid encoding Syncrip cDNA with a Myc-tag (RC217902, Origene) or a control plasmid (pcDNA3.1-eGFP, Addgene).

Techniques: Western Blot, In Vitro, Incubation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Post transcriptional regulation of HLA-A protein expression by alternative polyadenylation signals involving the RNA binding protein Syncrip

doi: 10.4049/jimmunol.1700697

Figure Lengend Snippet: RNA immunoprecipitation was carried out to confirm differential binding of Syncrip to the long 3’UTR of cellular HLA-A mRNA. (A) Transformed B cell lines from individuals with HLA-A*11 or HLA-A*03 homozygous genotypes were transfected with a c-Myc tagged Syncrip encoding vector (Syncrip-Myc) or with pcDNA3-GFP (Control). Cells were lysed after 48 hours and expression of c-Myc tagged Syncrip as well as GFP-control was confirmed using Western blot. (B) RNA immunoprecipitation was performed with the anti-c-Myc antibody coated magnetic beads. Specific pull down of myc-tagged Syncrip was confirmed by Western blot. (C) Fold enrichment of HLA-A 3’UTR in the immunoprecipitated RNA was analyzed by qPCR. (D) EBV transformed B cell lines from individuals with HLA-A*11 or HLA*03 homozygous genotypes were transfected with a Syncrip targeting siRNA and cultured for 72 hours. Syncrip knockdown was confirmed by Western blot. (E) Cell surface expression levels of the HLA-A allotypes treated with the Syncrip siRNA (open black curve) or a control siRNA (gray curve) were measured using allele specific antibodies. Cell surface expression of HLA-A*03 (long form of 3’UTR) was enhanced on the cells transfected with Syncrip targeting siRNA, whereas expression of HLA-A*11 (short form of 3’UTR) remained unaffected. The dotted curves indicate isotype control. Fold change in expression levels was calculated as the ratio of mean fluorescence intensity (MFI) of HLA-A vs isotype control. A histogram of one of three comparable experiments performed is shown for each allele. The mean ±SE (n=3) are depicted as horizontal and vertical bars for each group, respectively. Student t tests were used for statistical comparisons and two tailed p value is indicated.

Article Snippet: EBV transformed B lymphoblastoid cell lines derived from individuals with known HLA-A genotypes ( HLA-A*11 or A*03 homozygotes) were transfected with a plasmid encoding Syncrip cDNA with a Myc-tag (RC217902, Origene) or a control plasmid (pcDNA3.1-eGFP, Addgene).

Techniques: RNA Immunoprecipitation, Binding Assay, Transformation Assay, Transfection, Plasmid Preparation, Control, Expressing, Western Blot, Magnetic Beads, Immunoprecipitation, Cell Culture, Knockdown, Fluorescence, Two Tailed Test