rbpj  (Proteintech)

Journal: Poultry Science

Article Title: Multi-omics analysis reveals RBPJ-mediated regulation of EGF/ACTN2 / MYPN / COL21A1 in fibroblast during oviduct functional remodeling of duck

doi: 10.1016/j.psj.2026.106688

Figure Lengend Snippet: Identification of duck embryo fibroblasts and the effects of RBPJ overexpression on proliferation. (A) Immunofluorescence identification of decorin and vimentin in duck embryo fibroblasts. Target proteins (red, Alexa Fluor 488), nuclei (blue, Hochest 33342). Scale bar =100 μm. (B) Relative expression level of RBPJ mRNA, plot with Control as 1. (C) GAPDH and RBPJ protein band diagrams. (D) Quantified relative protein bands, plot with Control as 1. (E) Cell viability curve after RBPJ overexpression. (F) Staining maps of Group C and OE. (G) Quantitative plot of the proportion of EdU positive cells in two groups. pCD3.1 is the plasmid of group C and pCD3.1-RBPJ is the plasmid of group OE of RBPJ ( ⁎⁎ P < 0.01; ⁎⁎⁎ P < 0.001; ⁎⁎⁎⁎ P < 0.0001).

Article Snippet: The membrane was then incubated overnight at 4°C with the following primary antibodies diluted in Primary Antibody Dilution Buffer (P0023A, Beyotime, China): RBPJ (mouse monoclonal antibody, 1:2000, 66132-1-Ig, Proteintech, China), glyceraldehyde-3-phosphate dehydrogenase ( GAPDH , rabbit polyclonal antibody, 1:1000, 10494-1-AP, Proteintech, China) and β-tubulin (mouse monoclonal antibody,1:10000, 66240-1-Ig, Proteintech, China) (GAPDH and β-tubulin was used as the internal reference gene for normalization).

Techniques: Over Expression, Immunofluorescence, Expressing, Control, Staining, Plasmid Preparation

Journal: mBio

Article Title: HCMV promotes viral reactivation through the coordinated regulation of Notch signaling by UL8 and miR-UL36

doi: 10.1128/mbio.03377-25

Figure Lengend Snippet: miR-UL36 impairs Notch signaling through targeting Notch3 and RBPJ. ( A ) HEK293 cells were transfected with a 4× CSL-Luciferase construct as well as a plasmid expressing the intracellular domain of Notch3 and either negative control miRNA mimic, miR-US33, miR-UL36, or miR-UL112. Protein lysates were harvested 24 h post-transfection, and luciferase expression was monitored using a luminometer ( n = 3; P < 0.05 calculated using a one-sample t and Wilcoxon test). ( B and C ) NHDFs were transfected with negative control miRNA mimic, miR-UL36, or siRNA against Notch3 ( B ) or RBPJ ( C ). Protein lysates were harvested 48 h post-transfection, and immunoblots were performed using the indicated antibodies. Densitometry was performed using ImageJ software for three independent experiments and plotted in the right-hand panels. ( D and E ). NHDF were mock-infected or infected with WT HCMV or a miR-UL36 mutant virus for 48 h. Protein lysates were harvested, and immunoblotting was performed using the indicated antibodies. Densitometry was performed using ImageJ software for three independent experiments and plotted in the right-hand panels. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 as determined by two-tailed t -test. ( F ) RNA was harvested from cells infected as in ( D and E ) and subjected to qRT-PCR using primers for the Notch target genes HES1 and HEY1. ( n = 3; * P < 0.05 by two-tailed t -test).

Article Snippet: The following commercial antibodies were used: Notch3 (2889, Cell Signaling), RBPJ (5442, Cell Signaling), GAPDH (ab8245, Abcam), HCMV IE2 (MAB810, Sigma Aldrich), UL44 (Virusys Corporation, P1202-2), and Turbo ID (AS204440, Agrisera).

Techniques: Transfection, Luciferase, Construct, Plasmid Preparation, Expressing, Negative Control, Western Blot, Software, Infection, Mutagenesis, Virus, Two Tailed Test, Quantitative RT-PCR

Journal: MedComm

Article Title: Nitrate Enhances Gastric Mucosa Defense and Repair Process in Ethanol‐Induced Gastric Ulcer Rats via the Notch–Tff2 Pathway

doi: 10.1002/mco2.70628

Figure Lengend Snippet: Nitrate functions by Notch pathway inhibition positively transcripting TFF2 in vitro. (A) Diagram of the wild‐type and mutant sequences for the three predicted RBPJ binding sites within the 251 bp TFF2 promoter probes. (B) Representative electrophoretic mobility shift assay (EMSA) autoradiograph. Hot probe is the biotin‐labeled wild‐type oligonucleotides of the truncated TFF2 promoter containing the binding motif; Mutant probe is the labeled oligonucleotides sequence with nucleotides mutated. The cold probe is nonlabeled competitive wild‐type probes (100 and 50 that of the concentrations). The shifted bands are indicated by arrows, which suggested the formation of DNA–protein complexes (lane 2, 3, 7). The super shifted bands indicated the formation of DNA–protein–antibody complexes (lane 3, 7). “+” and “−” represent presence and absence, respectively. (C) Relative TFF2 promoter (Full, Mut1, Mut2, and Mut3) luciferase activity was detected by DLR assays in RBPJ overexpressed and normal‐expressed GES‐1 cells. (D) A schematic diagram showing the location of RBPJ putative binding regions on the TFF2 promoter. (E) RT‐qPCR analysis of TFF2 binding site expression of GES‐1 cells. Target site expression in RBPJ‐treated groups was normalized to IgG negative control groups and expressed as fold change relative to the IgG groups. (F) IF staining of NICD (pink) and DAPI (blue). Scale bar = 50 µm. (G) IF analysis of MFI of nuclear NICD in positive cells. (H) Representative immunoblotting band of Notch signaling pathway in GES‐1 cells. (I–K) Analyses of immunoblotting band gray value of (H). (L) RT‐qPCR analysis of TFF2 mRNA expression of DMSO/DAPT treated GES‐1 cells. Target gene expression was normalized to GAPDH mRNA and expressed as fold change relative to the DMSO vehicle group. (M) RT‐qPCR analysis of TFF2 mRNA expression in RBPJ overexpressed and NICD deprived GES‐1 cells. The target gene expression was normalized to GAPDH mRNA and expressed as fold change relative to the vector + DMSO group. (N) RT‐qPCR analysis of TFF2 mRNA expression in RBPJ overexpressed and NICD‐RBPJ overexpressed GES‐1 cells. The target gene expression was normalized to GAPDH mRNA and expressed as fold change relative to the vector1 + vector2 group. (O) Representative image of PLA of NICD–RBPJ proximity. Each red dot represents a positive signal of NICD–RBPJ interaction and nuclei were counterstained with DAPI (blue). Scale bar = 20 µm. Quantitative data are expressed as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, and ns denotes no significance. Nit, nitrate; EtOH, ethanol; GES‐1, human gastric epithelial; NICD, intracellular structural domain; RBPJ, recombination signal binding protein for immunoglobulin kappa J region; EMSA, electrophoretic mobility shift assay; TFF2, trefoil factor 2; DAPI, 2‐(4‐amidinophenyl)‐6‐indolecarbamidine dihydrochloride; Yhhu‐3792, N2‐(4‐isopropylphenyl)‐5‐(3‐methoxyphenoxy) quinazoline‐2,4‐diamine; Luc, luciferase; SD, standard deviation; DLR, dual‐luciferase report; PLA, proximity ligation assay.

Article Snippet: The probes were incubated with the GES‐1 cells extract and RBPJ antibody (5313; Cell Signaling Technology, USA) at room temperature for 50 min. To comprehend the specificity of the DNA/protein binding reactions, competition assays were performed with 50/100 excessive unlabeled cold probes.

Techniques: Inhibition, In Vitro, Mutagenesis, Binding Assay, Electrophoretic Mobility Shift Assay, Autoradiography, Labeling, Sequencing, Luciferase, Activity Assay, Quantitative RT-PCR, Expressing, Negative Control, Staining, Western Blot, Targeted Gene Expression, Plasmid Preparation, Standard Deviation, Proximity Ligation Assay

Journal: bioRxiv

Article Title: Graded Notch Signaling Functions as a Rheostat of Lineage Plasticity and Therapy Resistance in Prostate Cancer

doi: 10.1101/2025.11.23.690056

Figure Lengend Snippet: (A, B) Relative gene expression of Notch pathway components and lineage markers in enzalutamide-resistant cell lines, (A) gTP53/RB1-R and (B) PC3, after treatment with a gamma-secretase inhibitor (DAPT), an RBPJ inhibitor (RIN1), or DMSO vehicle control. Significance is denoted as *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: DMSO (vehicle control), 10 μM DAPT (MedChemExpress, HY-13027), or 5 μM RIN1 (MedChemExpress, HY-137471) were added to 3 biological replicates each.

Techniques: Gene Expression, Control