antibody rabbit anti nrbp2  (Proteintech)

Journal: Cancers

Article Title: Nuclear Receptor Binding Protein 2 Is Downregulated in Medulloblastoma, and Reduces Tumor Cell Survival upon Overexpression

doi: 10.3390/cancers12061483

Figure Lengend Snippet: Expression of nuclear receptor binding protein 2 (NRBP2) in human brain tumors. ( A ) Quantification of the NRBP2 positive staining area in human brain tumors; ( B ) Quantification of NRBP2 staining intensity in human brain tumors; ( C ) Bubble plot comparing the staining quantity (% positive staining) and intensity of NRBP2 in the nucleus (left) or cytoplasm (right) across all samples; ( D ) Immunohistochemistry staining of NRBP2 in non-tumor brain and medulloblastoma tissues of the same tissue microarray, scale bar 20 µm; ( E ) Top panel: NRBP2 protein expression (western blot) in fetal human cerebellum and in medulloblastoma cells D283, D324, MB002 and sD425, Bottom panel: Relative NRBP2 protein expression, normalized to b-actin as loading control of the western blot above, setting fetal human cerebellum (CB) = 1; ( F ) NRBP2 gene expression levels (GSE107405) in MB cell lines (D283, D324, MB002, sD425) in triplicates, and single samples of human cerebellar astrocytes (HA-c) and human spinal cord astrocytes (HA-sp); ( G ) NRBP2 is mainly present in the cytoplasm as seen by subcellular fractionation followed by western blot of cell lysates from a central nervous system(CNS) embryonal tumor cell line PFSK, MB cell lines D283 and D324, and glioblastoma cells U3013MG (Left panel) and the corresponding quantification of intensity (Right panel).

Article Snippet: The membrane was blocked in 5% non-fat milk in TBST (Tris buffered saline supplemented with Tween-20)for 1 h at room temperature and then incubated with primary antibody rabbit anti-NRBP2 (1:1000, Proteintech, Manchester, UK), p-AKT pSer473 (1:000, Cell Signaling Technology, Danvers, MA, USA), cleaved caspase 3 (1:1000, Cell Signaling, MA, USA), mouse monoclonal BAK1 (1:500, Santa Cruz Biotechnology, Dallas, TX, USA), mouse monoclonal Bax (1:500, Santa Cruz Biotechnology), β-Actin (1:500, Santa Cruz Biotechnology), mouse monoclonal Ki67 (1:400, Santa Cruz Biotechnology) overnight at 4 °C.

Techniques: Expressing, Binding Assay, Staining, Immunohistochemistry, Microarray, Western Blot, Fractionation

Journal: Cancers

Article Title: Nuclear Receptor Binding Protein 2 Is Downregulated in Medulloblastoma, and Reduces Tumor Cell Survival upon Overexpression

doi: 10.3390/cancers12061483

Figure Lengend Snippet: Expression of nuclear receptor binding protein (NRBP2) in human medulloblastoma. ( A ) NRBP2 gene expression levels (GSE124814) in normal human cerebellum, either for pediatric cases only (age < 18 years; n = 47) or across pediatric and adult cases (all ages; n = 291), and MB patients ( n = 1350); ( B ) NRBP2 gene expression levels (GSE85217) in wingless (WNT) ( n = 70), sonic hedgehog (SHH) ( n = 223), Group 3 ( n = 144), and Group 4 ( n = 326) subgroups of medulloblastoma patients. Boxes indicate the range between the first and third quartiles, black horizontal lines represent the median value, and whiskers extend to the extreme values excluding outliers, which are shown as dots; ( C ) NRBP2 protein expression levels (MSV00008264) in WNT ( n = 3), SHH ( n = 15), Group 3 ( n = 14) and Group 4 ( n = 13) subgroups of MB patients. Boxes indicate the range between 1–99 percentile, black horizontal lines represent the median value, and whiskers extend to the extreme values. Unpaired t test between the SHH and G3 groups showed a significant difference (* denotes significance as p < 0.05).

Article Snippet: The membrane was blocked in 5% non-fat milk in TBST (Tris buffered saline supplemented with Tween-20)for 1 h at room temperature and then incubated with primary antibody rabbit anti-NRBP2 (1:1000, Proteintech, Manchester, UK), p-AKT pSer473 (1:000, Cell Signaling Technology, Danvers, MA, USA), cleaved caspase 3 (1:1000, Cell Signaling, MA, USA), mouse monoclonal BAK1 (1:500, Santa Cruz Biotechnology, Dallas, TX, USA), mouse monoclonal Bax (1:500, Santa Cruz Biotechnology), β-Actin (1:500, Santa Cruz Biotechnology), mouse monoclonal Ki67 (1:400, Santa Cruz Biotechnology) overnight at 4 °C.

Techniques: Expressing, Binding Assay

Journal: Cancers

Article Title: Nuclear Receptor Binding Protein 2 Is Downregulated in Medulloblastoma, and Reduces Tumor Cell Survival upon Overexpression

doi: 10.3390/cancers12061483

Figure Lengend Snippet: Effects on nuclear receptor binding protein (NRBP2) mRNA expression and cell growth of medulloblastoma (MB) cell lines by promotor de-methylation and histone de-acetylation treatment. ( A ) relative NRBP2 expression in D283, D324, sD425, and MB002 cell lines treated with 5-Aza-2′-deoxycytidine (dAC) for two days, expressed as RQ value, 2-(ΔΔCt) value, beta-actin was used as an endogenous control and untreated cells as the reference. n = 3; ( B ) relative NRBP2 expression in D283, D324, sD425, and MB002 cell lines treated with Valproic acid (VPA) for two days, expressed as RQ (relative quantification) value, 2-(ΔΔCt) value, beta-actin was used as an endogenous control and untreated cells as the reference. n = 3; ( C ) Left Upper panel: NRBP2 protein expression in MB002, sD425, and D324 cell lines treated with dAC for two days. Left Lower panel: Signal intensity quantification by ImageJ. Right Upper panel: NRBP2 protein expression in MB002, sD425 and D324 cell lines treated with VPA for two days. Right Lower panel: Signal intensity quantification by ImageJ (an open source image processing program developed at the National Institute of Health, USA) . b-actin was used as a loading control for both; ( D ) Left panel: growth curves for MB002 (upper) and sD425 (lower) cell lines treated with VPA compared to untreated cells. Right panel: growth curves for MB002 (upper) and sD425 (lower) cell lines treated with dAC compared to untreated cells. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ( E ) Left panel: relative gene expression levels of NCOR after transfection by off-target-siRNA, NCOR-siRNA, SMRT-siRNA, and NCOR and SMRT-siRNA; middle panel: relative gene expression levels of SMRT after treatment by off-target-siRNA, NCOR-siRNA, SMRT-siRNA, and NCOR and SMRT-siRNA; right panel: relative gene expression levels of NRBP2 after treatment by off-target-siRNA, NCOR-siRNA, SMRT-siRNA, and NCOR and SMRT-siRNA. n = 3. Average expression levels after off-target-siRNA transfection were used as control.

Article Snippet: The membrane was blocked in 5% non-fat milk in TBST (Tris buffered saline supplemented with Tween-20)for 1 h at room temperature and then incubated with primary antibody rabbit anti-NRBP2 (1:1000, Proteintech, Manchester, UK), p-AKT pSer473 (1:000, Cell Signaling Technology, Danvers, MA, USA), cleaved caspase 3 (1:1000, Cell Signaling, MA, USA), mouse monoclonal BAK1 (1:500, Santa Cruz Biotechnology, Dallas, TX, USA), mouse monoclonal Bax (1:500, Santa Cruz Biotechnology), β-Actin (1:500, Santa Cruz Biotechnology), mouse monoclonal Ki67 (1:400, Santa Cruz Biotechnology) overnight at 4 °C.

Techniques: Binding Assay, Expressing, Methylation, Transfection

Journal: Cancers

Article Title: Nuclear Receptor Binding Protein 2 Is Downregulated in Medulloblastoma, and Reduces Tumor Cell Survival upon Overexpression

doi: 10.3390/cancers12061483

Figure Lengend Snippet: Overexpression of nuclear receptor binding protein (NRBP2) in D324 medulloblastoma (MB) cells causes reduction of cell numbers, migration, and invasion. ( A ) Western blot of NRBP2 in D324 cells transfected with an empty vector (cntl) or NRBP2-IRES2-eGFP NRBP2 sequence with V5 tag and linked with the internal ribosome entry site (IRES2) and the enhanced green fluorescent protein (eGFP) coding region) overexpression plasmid, Left panel: Signal intensity quantification by ImageJ; ( B ) growth rate of D324 cells transfected with control vector or NRBP2 vector; ( C ) Western blot of NRBP2 in D324 transduced with scrambled shRNA, or shRNA against NRBP2. The cultures were transduced twice to increase the efficiency of downregulation, Left panel: Signal intensity quantification by ImageJ; ( D ) growth curve of D324 cells transduced with scrambled shRNA, or shRNA against NRBP2; ( E ) photomicrographs of cell migration after scratching the cell monolayer ( n = 3); ( F ) quantification of coverage of cell-free area from ( C ) ( n = 3); ( G ) photomicrographs of cell invasion in collagen gel at different time points of D324 cells transduced with scrambled shRNA, or shRNA against NRBP2; ( H ) quantification of invasion from ( E ) ( n = 3). * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: The membrane was blocked in 5% non-fat milk in TBST (Tris buffered saline supplemented with Tween-20)for 1 h at room temperature and then incubated with primary antibody rabbit anti-NRBP2 (1:1000, Proteintech, Manchester, UK), p-AKT pSer473 (1:000, Cell Signaling Technology, Danvers, MA, USA), cleaved caspase 3 (1:1000, Cell Signaling, MA, USA), mouse monoclonal BAK1 (1:500, Santa Cruz Biotechnology, Dallas, TX, USA), mouse monoclonal Bax (1:500, Santa Cruz Biotechnology), β-Actin (1:500, Santa Cruz Biotechnology), mouse monoclonal Ki67 (1:400, Santa Cruz Biotechnology) overnight at 4 °C.

Techniques: Over Expression, Binding Assay, Migration, Western Blot, Transfection, Plasmid Preparation, Sequencing, Transduction, shRNA

Journal: Cancers

Article Title: Nuclear Receptor Binding Protein 2 Is Downregulated in Medulloblastoma, and Reduces Tumor Cell Survival upon Overexpression

doi: 10.3390/cancers12061483

Figure Lengend Snippet: Overexpression of nuclear receptor binding protein (NRBP2) in medulloblastoma (MB) leads to increased cell death. ( A ) flow-cytometry-based quantification of live and dead D324 cells 1 and 2 days post-transfection with NRBP2 -V5-IRES-eGFP (NRBP2 sequence with V5 tag, linked with the internal ribosome entry site (IRES2) and the enhanced green fluorescent protein (eGFP) coding region) plasmid; ( B ) flow-cytometry-based cell-cycle analysis in D324 cells after transfection with NRBP2 -V5-IRES-eGFP plasmid; ( C ) Western blot of Ki67 in MB002 and sD425 cells 2–24 h post transduction with empty vector or EF1A-NRBP2(V5)-plasmid. Corresponding Signal intensity quantification by ImageJ on the left; ( D ) Western blot of phosphorylation of AKT in D324 cells transfected with control or NRBP2 plasmid and signal intensity quantification by ImageJ on the left; ( E ) apoptosis analysis based on Annexin V labelling of control-transfected or NRBP2-transfected D324 cells; ( F ) Western blot of cleaved caspase-3 in MB002 and sD425 cells transduced with empty vector or NRBP2 overexpressing plasmid, 48 h post transduction; ( G ) cytospin preparations of MB002 cells, 4 to 48 h after transduction with a EF1A-NRBP2(V5)- plasmid showing staining for NRBP2 (upper panel) and cleaved caspase-3 (lower panel); ( H ) expression of apoptotic genes BAK1 and BAX mRNA in D324 cells transfected with control or NRBP2 plasmid; ( I ) Western blot of BAK1 and BAX in MB002 and sD425 cells transduced with control or NRBP2 plasmid 48 h post transduction. Corresponding signal intensity quantification by ImageJ is shown on the left.

Article Snippet: The membrane was blocked in 5% non-fat milk in TBST (Tris buffered saline supplemented with Tween-20)for 1 h at room temperature and then incubated with primary antibody rabbit anti-NRBP2 (1:1000, Proteintech, Manchester, UK), p-AKT pSer473 (1:000, Cell Signaling Technology, Danvers, MA, USA), cleaved caspase 3 (1:1000, Cell Signaling, MA, USA), mouse monoclonal BAK1 (1:500, Santa Cruz Biotechnology, Dallas, TX, USA), mouse monoclonal Bax (1:500, Santa Cruz Biotechnology), β-Actin (1:500, Santa Cruz Biotechnology), mouse monoclonal Ki67 (1:400, Santa Cruz Biotechnology) overnight at 4 °C.

Techniques: Over Expression, Binding Assay, Flow Cytometry, Transfection, Sequencing, Plasmid Preparation, Cell Cycle Assay, Western Blot, Transduction, Staining, Expressing

Journal: Cancers

Article Title: Nuclear Receptor Binding Protein 2 Is Downregulated in Medulloblastoma, and Reduces Tumor Cell Survival upon Overexpression

doi: 10.3390/cancers12061483

Figure Lengend Snippet: List of sequences of the primers used for qPCR.

Article Snippet: The membrane was blocked in 5% non-fat milk in TBST (Tris buffered saline supplemented with Tween-20)for 1 h at room temperature and then incubated with primary antibody rabbit anti-NRBP2 (1:1000, Proteintech, Manchester, UK), p-AKT pSer473 (1:000, Cell Signaling Technology, Danvers, MA, USA), cleaved caspase 3 (1:1000, Cell Signaling, MA, USA), mouse monoclonal BAK1 (1:500, Santa Cruz Biotechnology, Dallas, TX, USA), mouse monoclonal Bax (1:500, Santa Cruz Biotechnology), β-Actin (1:500, Santa Cruz Biotechnology), mouse monoclonal Ki67 (1:400, Santa Cruz Biotechnology) overnight at 4 °C.

Techniques:

Journal: PLoS ONE

Article Title: The altered expression of autophagy-related genes participates in heart failure: NRBP2 and CALCOCO2 are associated with left ventricular dysfunction parameters in human dilated cardiomyopathy

doi: 10.1371/journal.pone.0215818

Figure Lengend Snippet: Bar graphs comparing the NRBP2 (A) and CALCOCO2 (B) protein levels in dilated hearts (gray bars) vs . control (CNT) hearts (black bars). The values from the CNTs were set to 1. Bars display the fold change ± standard error of the mean. * P < 0.001 vs . CNTs.

Article Snippet: The primary antibodies used were anti-NRBP2 rabbit polyclonal antibody (1:2000; ab227480) and anti-CALCOCO2 rabbit polyclonal antibody (1:800; ab68588), both obtained from Abcam (Cambridge, UK); the mouse monoclonal anti-GAPDH antibody (1:800; ab-9484) from Abcam (Cambridge, UK) was used as a loading control.

Techniques: