Journal: Disease Markers

Article Title: Differential Gene Expression Profile of Renin-Angiotensin System in the Left Atrium in Mitral Regurgitation Patients

doi: 10.1155/2018/6924608

Figure Lengend Snippet: TaqMan real-time PCR assay identification.

Article Snippet: LNPEP , Hs00893646_m1.

Techniques: Real-time Polymerase Chain Reaction

Journal: Disease Markers

Article Title: Differential Gene Expression Profile of Renin-Angiotensin System in the Left Atrium in Mitral Regurgitation Patients

doi: 10.1155/2018/6924608

Figure Lengend Snippet: Comparison of mRNA levels through quantitative PCR in the left atria among MR patients with heart failure, aortic valve disease patients with heart failure, and normal controls.

Article Snippet: LNPEP , Hs00893646_m1.

Techniques: Comparison, Real-time Polymerase Chain Reaction

Journal: Disease Markers

Article Title: Differential Gene Expression Profile of Renin-Angiotensin System in the Left Atrium in Mitral Regurgitation Patients

doi: 10.1155/2018/6924608

Figure Lengend Snippet: Quantitative determination of mRNA of (a) cathepsin A ( CTSA ), (b) leucyl/cystinyl aminopeptidase ( LNPEP ), and (c) MAS1 oncogene ( MAS1 ) by real-time RT-PCR in the left atrium in mitral regurgitation (MR) patients with heart failure ( n = 10), in aortic valve disease patients (AVD) with heart failure ( n = 8), and in purchased samples from normal subjects (NC) ( n = 6). ∗ P < 0.05.

Article Snippet: LNPEP , Hs00893646_m1.

Techniques: Quantitative RT-PCR

Journal: Frontiers in Pharmacology

Article Title: The NLRP3 inflammasome is involved in resident intruder paradigm-induced aggressive behaviors in mice

doi: 10.3389/fphar.2023.974905

Figure Lengend Snippet: Behavioral phenotype of mice in each group. (A) Schematic representation of the experimental design. (B) Open field test representative trajectory graph. (C) Elevated plus-maze test representative trajectory graph. (D) Open field test results. (E) Elevated plus-maze test results. (F) Attack behavior test results. Data are expressed as means ± SEMs. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Control, producers of non-aggressive behavior after stress; Model, aggressive behavior after stress; A804598, P2X7R antagonist; IL-1Ra, IL-1β blocker; NLRP3 −/− , NLRP3 knockout mice. n = 8/group. (TIFF format, 1200 dpi, 2-column fitting image).

Article Snippet: Chemicals are listed as follows: A804598 (HY-100483, MedChemExpress Co., Ltd. United States); IL-1Ra (HY-P7029A, MCE, United States); DMSO (D8370, Solarbio, China); Mouse NLRP3 ELISA kit (JYM0765Mo, Wuhan ColorfulGene biological technology Co., Ltd. China); Mouse IL-1β ELISA kit (JYM0531Mo, Wuhan ColorfulGene biological technology Co., Ltd. China); BCA Protein Assay Kit (PC0020, Solarbio, China); SDS-PAGE Gel Rapid Preparation Kit (G2037-50T, Servicebio, China); Recombinant Anti-beta Actin antibody (GB15003, Servicebio, China); NLRP3 Antibody (DF7438, Affinity Biologicals, Canada); Recombinant Anti-P2X7 antibody (ab259942, abcam, United Kingdom); HRP-conjugated goat anti-rabbit IgG (GB23204, Servicebio, China); Anti-Iba1 Rabbit pAb (GB11105, Servicebio, China); Cy3-labeled goat anti-rabbit IgG (GB21303, Servicebio, China); FITC-labeled goat anti-rabbit IgG (GB22303, Servicebio, China); Anti-BrdU Mouse mAb (GB12051, Servicebio, China); Anti-NeuN Rabbit pAb (GB11138, Servicebio, China); HE staining solution (G1005-100ML, Servicebio, China).

Techniques: Control, Knock-Out

Journal: Frontiers in Pharmacology

Article Title: The NLRP3 inflammasome is involved in resident intruder paradigm-induced aggressive behaviors in mice

doi: 10.3389/fphar.2023.974905

Figure Lengend Snippet: The P2X7R was activated in the resident intruder paradigm. (A) Representative images of P2X7R (green) and DAPI (blue) labeling in the hippocampus CA1, CA3, and DG after drug interventions; scale bar = 200 and 50 µm. (B) Positive staining area of P2X7R and DAPI double-labeled (P2X7R + DAPI) proteins in the hippocampus CA1 (** p < 0.01 Control vs. Model ; n = 3/group), CA3 (** p < 0.01 Control vs. Model ; * p < 0.05 A804598 vs. Model ; n = 3/group), and DG (** p < 0.01 Control vs. Model ; * p < 0.05 A804598 vs. Model ; n = 3/group). (C) Representative immunoreactive bands showing the protein levels of hippocampal P2X7R in the Control, Model, A804598, IL-1Ra, and NLRP3 −/− mice. (D) Statistical results show that A804598, IL-1Ra, and NLRP3 −/− decreased the protein expression of P2X7R ( n = 3, **** p < 0.0001 vs. Model).

Article Snippet: Chemicals are listed as follows: A804598 (HY-100483, MedChemExpress Co., Ltd. United States); IL-1Ra (HY-P7029A, MCE, United States); DMSO (D8370, Solarbio, China); Mouse NLRP3 ELISA kit (JYM0765Mo, Wuhan ColorfulGene biological technology Co., Ltd. China); Mouse IL-1β ELISA kit (JYM0531Mo, Wuhan ColorfulGene biological technology Co., Ltd. China); BCA Protein Assay Kit (PC0020, Solarbio, China); SDS-PAGE Gel Rapid Preparation Kit (G2037-50T, Servicebio, China); Recombinant Anti-beta Actin antibody (GB15003, Servicebio, China); NLRP3 Antibody (DF7438, Affinity Biologicals, Canada); Recombinant Anti-P2X7 antibody (ab259942, abcam, United Kingdom); HRP-conjugated goat anti-rabbit IgG (GB23204, Servicebio, China); Anti-Iba1 Rabbit pAb (GB11105, Servicebio, China); Cy3-labeled goat anti-rabbit IgG (GB21303, Servicebio, China); FITC-labeled goat anti-rabbit IgG (GB22303, Servicebio, China); Anti-BrdU Mouse mAb (GB12051, Servicebio, China); Anti-NeuN Rabbit pAb (GB11138, Servicebio, China); HE staining solution (G1005-100ML, Servicebio, China).

Techniques: Labeling, Staining, Control, Expressing

Journal: Frontiers in Pharmacology

Article Title: The NLRP3 inflammasome is involved in resident intruder paradigm-induced aggressive behaviors in mice

doi: 10.3389/fphar.2023.974905

Figure Lengend Snippet: Correlations between the expression levels of NLRP3 in the hippocampus or serum and aggressive behaviors. (A) Representative immunoreactive bands showing the protein levels of hippocampal NLRP3 in the Control, Model, A804598, IL-1Ra, and NLRP3 −/− mice. (B) Statistical results show that A804598, IL-1Ra, and NLRP3 −/− decreased the protein expression of NLRP3 ( n = 3, **** p < 0.0001 vs. Model). (C) NLRP3 and IL-1βhippocampus and serum levels. (D) Pearson correlation analyses were performed by comparing the expression levels of NLRP3 in the hippocampus or serum and aggressive behavior latency or aggressive behavior score. Correlations were performed considering some animals ( N = 3). The coefficient r and p -value for each correlation are presented in a box. Statistically significant correlations are highlighted in red ( p < 0.05). Data are expressed as means ± SEMs. Control, producers of non-aggressive behavior after stress; Model, aggressive behavior after stress; A804598, P2X7R antagonist; IL-1Ra, IL-1β blocker; NLRP3 −/− , NLRP3 knockout mice. (TIFF format, 1200 dpi, 2-column fitting image).

Article Snippet: Chemicals are listed as follows: A804598 (HY-100483, MedChemExpress Co., Ltd. United States); IL-1Ra (HY-P7029A, MCE, United States); DMSO (D8370, Solarbio, China); Mouse NLRP3 ELISA kit (JYM0765Mo, Wuhan ColorfulGene biological technology Co., Ltd. China); Mouse IL-1β ELISA kit (JYM0531Mo, Wuhan ColorfulGene biological technology Co., Ltd. China); BCA Protein Assay Kit (PC0020, Solarbio, China); SDS-PAGE Gel Rapid Preparation Kit (G2037-50T, Servicebio, China); Recombinant Anti-beta Actin antibody (GB15003, Servicebio, China); NLRP3 Antibody (DF7438, Affinity Biologicals, Canada); Recombinant Anti-P2X7 antibody (ab259942, abcam, United Kingdom); HRP-conjugated goat anti-rabbit IgG (GB23204, Servicebio, China); Anti-Iba1 Rabbit pAb (GB11105, Servicebio, China); Cy3-labeled goat anti-rabbit IgG (GB21303, Servicebio, China); FITC-labeled goat anti-rabbit IgG (GB22303, Servicebio, China); Anti-BrdU Mouse mAb (GB12051, Servicebio, China); Anti-NeuN Rabbit pAb (GB11138, Servicebio, China); HE staining solution (G1005-100ML, Servicebio, China).

Techniques: Expressing, Control, Knock-Out

Journal: Scientific Reports

Article Title: Development of a sandwich ELISA to detect circulating, soluble IRAP as a potential disease biomarker

doi: 10.1038/s41598-023-44038-1

Figure Lengend Snippet: The C-terminal domain of IRAP can be secreted into the circulation/extracellular milieu. IRAP contains two functional domains: a cytosolic N-terminal domain that contains trafficking motifs and an extracellular/intra-luminal C-terminal domain that contains the catalytic site. Following cleavage at a site near the transmembrane region, the C-terminal domain can be secreted. Current anti-IRAP antibodies that are commercially available only target the N-terminal domain.

Article Snippet: Following transfer, membranes were washed briefly in Tris-buffered saline-tween (TBS-T; 0.1% Tween-20 in 1 × TBS) and then placed on a shaker (70 rpm) in blocking buffer (5% skim milk/TBS-T) for 1 h. The rabbit anti-IRAP antibody (1:2000; Cell Signalling #6918) or the mouse anti-IRAP antibodies (0.0005 μg/ml) were diluted in the same blocking buffer to the appropriate concentration and incubated on membranes overnight on a shaker (70 rpm) at 4 °C.

Techniques: Functional Assay

Journal: Scientific Reports

Article Title: Development of a sandwich ELISA to detect circulating, soluble IRAP as a potential disease biomarker

doi: 10.1038/s41598-023-44038-1

Figure Lengend Snippet: The novel anti-IRAP antibodies can specifically detect human IRAP. ( a ) Representative Western blots (uncropped 12 lanes in same gel) showing binding of the novel mouse anti-IRAP antibodies (RB9, RF7, RH3, RG4; 0.5 µg/µl) to (1) full length IRAP derived from membrane preparations of HEK293T cells transiently overexpressing human IRAP (0.01 µg protein/µl) or (2) purified soluble human IRAP (0.001 µg/µl). The lanes for each antibody were imaged at either different exposure times or at the same exposure time across all antibodies (n = 3). Original, uncropped blots are provided in Supplementary Fig. . ( b ) The mouse anti-IRAP antibodies (1 µg/well) and the commercially available rabbit anti-IRAP antibody (Rb; 1:500 concentration) were tested for binding to mIRAP (10 µg protein/well) or purified sIRAP (0.001 µg/well) using an indirect ELISA. Data presented is the mean from two separate experiments performed in duplicate (n = 2). ( c ) The mouse RB9 clone (1 µg/well; red) and the rabbit antibody (1:500; purple) were tested against decreasing concentrations of purified sIRAP (0.001–0.01 µg/well) and mIRAP (10 µg protein/well). Simple linear regression was conducted (R 2 = 0.94). Data presented is the mean of absorbance readings from one experiment performed in duplicate (n = 1).

Article Snippet: Following transfer, membranes were washed briefly in Tris-buffered saline-tween (TBS-T; 0.1% Tween-20 in 1 × TBS) and then placed on a shaker (70 rpm) in blocking buffer (5% skim milk/TBS-T) for 1 h. The rabbit anti-IRAP antibody (1:2000; Cell Signalling #6918) or the mouse anti-IRAP antibodies (0.0005 μg/ml) were diluted in the same blocking buffer to the appropriate concentration and incubated on membranes overnight on a shaker (70 rpm) at 4 °C.

Techniques: Western Blot, Binding Assay, Derivative Assay, Membrane, Purification, Concentration Assay, Indirect ELISA

Journal: Scientific Reports

Article Title: Development of a sandwich ELISA to detect circulating, soluble IRAP as a potential disease biomarker

doi: 10.1038/s41598-023-44038-1

Figure Lengend Snippet: Specificity of the subcloned antibodies for IRAP. Western blots of the untagged and biotinylated newly subcloned monoclonal anti-IRAP antibodies RF7, RB9, RH3 and RG4 (0.0005 µg/µl) against either ( a ) purified soluble human IRAP (S; 0.001 µg/well) and full length human IRAP enriched membrane preparations of HEK293T cells transiently transfected with human IRAP cDNA (M; 0.1 µg proteins/well) or ( b ) protein lysate from cardiac tissue of 10-week old male IRAP KO or WT mice (20 µg protein/well), n = 1. All blots are imaged for the same exposure time. Original, uncropped blots are provided in Supplementary Fig. .

Article Snippet: Following transfer, membranes were washed briefly in Tris-buffered saline-tween (TBS-T; 0.1% Tween-20 in 1 × TBS) and then placed on a shaker (70 rpm) in blocking buffer (5% skim milk/TBS-T) for 1 h. The rabbit anti-IRAP antibody (1:2000; Cell Signalling #6918) or the mouse anti-IRAP antibodies (0.0005 μg/ml) were diluted in the same blocking buffer to the appropriate concentration and incubated on membranes overnight on a shaker (70 rpm) at 4 °C.

Techniques: Western Blot, Purification, Membrane, Transfection

Journal: Scientific Reports

Article Title: Development of a sandwich ELISA to detect circulating, soluble IRAP as a potential disease biomarker

doi: 10.1038/s41598-023-44038-1

Figure Lengend Snippet: Detection of increases in sIRAP expression in human plasma throughout the later stages of pregnancy. ( a ) Representative Western blot and quantification of the optical density (OD) of sIRAP (140–150 kDa) in plasma from healthy controls and women at later stages of pregnancy (28- & 36-weeks and full term; diluted 1:10), detected using the subcloned RF7 anti-IRAP antibody (0.0005 µg/µl). Control plasma is from three different participants and pregnant plasma is three different batches of pooled samples (n = 3). Data is expressed as mean ± SEM, was corrected for total protein concentration and analysed using a one-way ANOVA with Tukey’s post-hoc test, * p < 0.05, ** p < 0.01 compared to control. ( b ) Sandwich ELISAs using RF7-RB9-B (red) and RF7-RG4-B (green) antibody combinations was conducted to measure concentrations of IRAP in plasma from control and pregnant women (28- & 36-weeks & term) using a purified sIRAP standard curve (µg/ml). All samples were diluted 1:5. Data was analysed using simple linear regression analysis to interpolate the concentrations of each sample and a one-way ANOVA with Tukeys’s post-hoc test to compare groups to the control, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs control, n = 5–6. All data was corrected for the negative control and is presented as mean ± SEM.

Article Snippet: Following transfer, membranes were washed briefly in Tris-buffered saline-tween (TBS-T; 0.1% Tween-20 in 1 × TBS) and then placed on a shaker (70 rpm) in blocking buffer (5% skim milk/TBS-T) for 1 h. The rabbit anti-IRAP antibody (1:2000; Cell Signalling #6918) or the mouse anti-IRAP antibodies (0.0005 μg/ml) were diluted in the same blocking buffer to the appropriate concentration and incubated on membranes overnight on a shaker (70 rpm) at 4 °C.

Techniques: Expressing, Clinical Proteomics, Western Blot, Control, Protein Concentration, Purification, Negative Control

Journal: Journal of Clinical and Translational Hepatology

Article Title: IL1RA + Myeloid-derived Suppressor Cells Activate Epithelial-mesenchymal Transition to Facilitate Lymphatic and Hepatic Metastasis in Pancreatic Ductal Carcinoma

doi: 10.14218/JCTH.2025.00416

Figure Lengend Snippet: (A) tSNE plot showing the four neutrophil-derived MDSC subtypes. (B) The top five expressed genes of IL1RN + MDSCs in the four neutrophil-derived MDSC subsets. (C, D) Representative flow cytometry data and quantification graphs showing the proportion of IL1RA + MDSCs in LNN and LNP PDAC tissues. (E) Representative images of immunofluorescence staining of IL1RA + MDSCs in LNN and LNP PDAC tissues. Scale bar = 100 µm. (F) Trajectory of neutrophil-derived MDSCs along pseudotime in two-dimensional space. (G) Heatmap showing dynamic changes in gene expression along pseudotime. (H) Prognostic analysis of PDAC patients with low or high IL1RA + MDSC recruitment (stratified by the median IL1RA + MDSC proportion in all samples). Kaplan–Meier survival plots of 16 patients are shown. Data are presented as mean ± SD in (D). Statistical differences were assessed using a two-tailed Student’s t -test. LNN, lymph node-negative; LNP, lymph node-positive; PDAC, pancreatic ductal adenocarcinoma; MDSCs, myeloid-derived suppressor cells.

Article Snippet: Primary antibodies included CD11b (Abcam, Cambridge, UK), CD68 (Proteintech, Wuhan, China), MPO (Abcam), IL1RA (Proteintech), and VEGFA (Proteintech).

Techniques: Derivative Assay, Flow Cytometry, Immunofluorescence, Staining, Gene Expression, Two Tailed Test

Journal: Journal of Clinical and Translational Hepatology

Article Title: IL1RA + Myeloid-derived Suppressor Cells Activate Epithelial-mesenchymal Transition to Facilitate Lymphatic and Hepatic Metastasis in Pancreatic Ductal Carcinoma

doi: 10.14218/JCTH.2025.00416

Figure Lengend Snippet: (A) Schematic illustration of IL1RN + MDSC–PDAC co-culture system. (B) Transwell migration assays in PANC-1 and ASPC-1 cells treated with normal medium (Control), IL1RA + MDSC-conditioned medium (IL1RA + MDSC CM), or IL1RA + MDSC CM + Axitinib. Scale bar = 50 µm. (C) Western blot analysis of EMT markers (N-cadherin, E-cadherin, ZO-1, Claudin-1) in PANC-1 cells under treatments identical to (B); β-actin was detected as a control. Grayscale was determined using ImageJ. (D) Relative expression of stemness-related mRNA after treatment with IL1RA + MDSC CM or IL1RA + MDSC CM + Axitinib. (E) Representative images of subcutaneous tumors in BALB/c nude mice from the PDX model: Group 1 (Control, tumor tissue only); Group 2 (tumor tissue co-implanted with IL1RN + MDSCs); Group 3 (tumor tissue co-implanted with IL1RN + MDSCs + Axitinib). (F) Excised tumor tissues from (E). (G, H) Tumor weights (G) and volumes (H) from the mouse model presented as bar graphs. (I) Representative H&E staining images of mouse pancreatic tumor tissues. Scale bar = 100 µm. Data are represented as mean ± SD of three independent biological replicates in (B) and four independent replicates in (F and G). Statistical significance was determined by one-way ANOVA with Dunnett’s post-hoc test: ns p ≥ 0.05; * p < 0.05; *** p < 0.001; **** p < 0.0001. PDAC, pancreatic ductal adenocarcinoma; MDSCs, myeloid-derived suppressor cells.

Article Snippet: Primary antibodies included CD11b (Abcam, Cambridge, UK), CD68 (Proteintech, Wuhan, China), MPO (Abcam), IL1RA (Proteintech), and VEGFA (Proteintech).

Techniques: Co-Culture Assay, Migration, Control, Western Blot, Expressing, Staining, Derivative Assay