1d11 neutralizing antibody  (Bio X Cell)

Journal: Nature Communications

Article Title: Suppression of TGF-β/SMAD signaling by an inner nuclear membrane phosphatase complex

doi: 10.1038/s41467-025-58681-x

Figure Lengend Snippet: A Localization of endogenous SMAD2 in HeLa parental cells or lacking the indicated genes analyzed by immunofluorescence. DNA was labeled with 4′,6- diamidino- 2- phenylindole (DAPI). Scale bar: 10 μM. B Quantification of nuclear accumulation of SMAD2 from imaging experiments, as shown in ( A ), n = 3 independent experiments, p -values were indicated in the graph. One-way ANOVA (multiple comparison) was performed, and data are presented as mean values ± SD. C Levels of endogenous pSMAD2 and SMAD2 in HeLa parental cells or lacking the indicated genes. Cell lysates were analyzed by SDS-PAGE followed by immunoblotting with the indicated antibodies. GAPDH was used as a loading control. D Levels of endogenous pSMAD2 and SMAD2 in HeLa parental cells or CTDENP1 KO cells with indicated treatments. The 1D11 antibody against TGF-β cytokine was used at 30, 150, and 300 µg/mL; mouse IgG antibody was used at 300 µg/mL; the TGF-β receptor specific inhibitor SB431542 was used at 10 µM. Cell lysates were analyzed by SDS-PAGE followed by immunoblotting with the indicated antibodies. GAPDH was used as a loading control. E levels of p21, p15 transcripts in HeLa parental cells or lacking the indicated genes analyzed by RT-qPCR. n = 3 independent experiments, each with 3 technical replicates. p -values were indicated in the graph. One-way ANOVA (multiple comparison) was performed, and data are presented as mean values ± SD. F levels of endogenous p21 in HeLa parental cells or lacking the indicated genes. Cell lysates were analyzed by SDS-PAGE followed by immunoblotting with the indicated antibodies. GAPDH was used as a loading control. G Quantification of S phase cells assessed based on EdU incorporation and DAPI staining. At least 30000 cells were analyzed for each condition. n = 3 independent experiments, p -values were indicated in the graph. One-way ANOVA (multiple comparison) was performed, and data are presented as mean values ± SD. H The CTDNEP1-NEP1R1-MAN1 complex dephosphorylates and inactivates R-SMADs at the INM (see text for details).

Article Snippet: Inhibition of TGF-β ligands was performed with the neutralizing antibody, 1D11 (BioX-Cell # #BE0083), and isotype-matched IgG1 monoclonal control antibody (BioX-Cell #BE0057) were used at 30, 150, and 300 μg/mL, respectively, to treat HeLa parental cells or CTDENP1 KO cells with indicated time.

Techniques: Immunofluorescence, Labeling, Imaging, Comparison, SDS Page, Western Blot, Control, Quantitative RT-PCR, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Identification of a novel, MSC-induced macrophage subtype via single-cell sequencing: implications for intervertebral disc degeneration therapy

doi: 10.3389/fcell.2023.1286011

Figure Lengend Snippet: Comparative Analysis between Macrophages Cultured in Hypoxic Medium in the Presence and Absence of the TGF-β Neutralizing Antibody 1D11. (A) Macrophages were cultured in hypoxic medium and treated with TGF-β in the presence and absence of the TGF-β neutralizing antibody 1D11 and Western blotting was performed. (B–D) scRNA-Seq datasets from macrophages cultured in hypoxic MSC conditioned medium were integrated with macrophages cultured with the addition of the TGF-β neutralizing antibody 1D11 and comparative analyses were performed. (B) UMAP representation. The fraction (C) and (D) cells from each sample within each cluster are shown.

Article Snippet: To block effects of TGF-β isoforms, MSC conditioned media were incubated with the TGF-β isoform neutralizing antibody 1D11 (30 μg/mL, Thermo Fisher, #MA523795) for 2 h prior to use.

Techniques: Cell Culture, Western Blot

Journal: Military Medical Research

Article Title: Targeting GPR65 alleviates hepatic inflammation and fibrosis by suppressing the JNK and NF-κB pathways

doi: 10.1186/s40779-023-00494-4

Figure Lengend Snippet: GPR65 promotes HSCs activation indirectly by paracrine secretion of TGF-β from macrophage. The CM from control, GPR65-overexpressed, pH 6.6-treated or GPR65 agonist-treated macrophage cells, with 20 μg/ml TGF-β neutralization antibody 1D11 or mouse IgG, were used to treat primary HSCs for 24 h. qRT-PCR was used to assess the mRNA level of Gpr65 , Acta2 , Col1α1 , Col1α2 , Timp1 , Mmp2 and Tgfβ1 ( n = 3) ( a-c ); Western blotting was used to determine the protein level of COL1α1, α-SMA, MMP2 and TIMP1 ( d ); the expression and location of α-SMA and COL1α1 was assessed by confocal microscopy ( e ). Scale bar = 20 μm. qRT-PCR was used to assess the expression of Tgfβ1 and Tgfβ3 in GPR65-KO ( f ), Gpr65 -overexpressed ( g ), or GPR65 agonist/inhibitor ( h ) treated HMs ( n = 3). i TGF-β1 level in the supernatant of HMs transfected with pcDNA3.1 or pcDNA3.1-GPR65 was detected by ELISA ( n = 4). HMs isolated from WT and GPR65-KO mice were treated with or without GPR65 agonist ( j ) or pH 6.6 ( k ) for 24 h, TGF-β1 level in the supernatant was detected by ELISA ( n = 3). * P < 0.05 vs. pcDNA3.1, pH 7.4, DMSO, WT, or WT + DMSO/pH 7.4; # P < 0.05 vs. pcDNA3.1-GPR65, pH 6.6, GPR65 agonist, or WT + GPR65 agonist/pH 6.6. CM conditioned medium, ELISA enzyme-linked immunosorbent assay, HM hepatic macrophage, HSC hepatic stellate cell, KO knockout, qRT-PCR quantitative real-time reverse transcription-polymerase chain reaction, COL1α1 collagen type I alpha 1, α-SMA α-smooth muscle actin, MMP2 matrix metalloproteinase 2, TIMP1 tissue inhibitor of metalloproteinases 1, TGF-β1 transforming growth factor-β1

Article Snippet: The CM with 20 μg/ml transforming growth factor-β (TGF-β) neutralization antibody 1D11 (Thermo Fisher Scientific, USA, Cat# 16-9243-85; RRID: AB_2573124) or mouse IgG, was used to treat primary HSCs.

Techniques: Activation Assay, Neutralization, Quantitative RT-PCR, Western Blot, Expressing, Confocal Microscopy, Transfection, Enzyme-linked Immunosorbent Assay, Isolation, Knock-Out, Reverse Transcription Polymerase Chain Reaction

Journal: Military Medical Research

Article Title: Targeting GPR65 alleviates hepatic inflammation and fibrosis by suppressing the JNK and NF-κB pathways

doi: 10.1186/s40779-023-00494-4

Figure Lengend Snippet: Targeting GPR65 alleviates hepatic macrophage inflammation and fibrosis by suppressing the JNK/NF-κB pathways. a Western blotting was used to determine the level of p-IKK, p-IĸBα, p-NF-κB p65, IKK, IĸBα, NF-κB p65, p-MLK3, p-MKK4, p-MKK7, p-JNK, JNK, p-p44/42, p44/42, p-p38, p38, p-GSK-3β, p-Akt, Akt, p-PDK1, p-PTEN and p–c-Raf in liver tissues from WT, WT + BDL, GPR65-KO and GPR65-KO + BDL mice. b Western blotting was used to determine the level of p-IKK, IKK, p-IĸBα, IĸBα, p-p65, p65, p-MLK3, p-MKK7, p-JNK and JNK in GPR65-KO, GPR65-overexpressed, various pH-treated and GPR65 agonist/inhibitor-treated HMs. The specific inhibitors of JNK, SP600125 (10 μmol/L) and JNK-IN-8 (5 μmol/L), as well as the specific inhibitors of NF-κB, BAY 11–7082 (5 μmol/L) and APDC (20 μmol/L), were used to treat GPR65-overexpressed HMs for 24 h, and qRT-PCR was used to assess the expression of Tnfα , Il6 and Tgfβ3 ( n = 3) ( c ); TGF-β1 and IL-6 level in the supernatant were detected by ELISA ( n = 3) ( d ); the expression and location of TNF-α was assessed by confocal microscopy ( e ). Scale bar = 20 μm. * P < 0.05 vs. DMSO + pcDNA3.1; # P < 0.05 vs. DMSO + pcDNA3.1-GPR65. CCl 4 carbon tetrachloride, BDL bile duct ligation, ELISA enzyme-linked immunosorbent assay, HM hepatic macrophage, KO knockout, qRT-PCR quantitative real-time reverse transcription-polymerase chain reaction, IKK inhibitor of ĸB kinase, IĸBα inhibitor of ĸB α, NF-κB p65 nuclear factor κB p65 subunit, MLK3 mixed lineage kinase 3, MKK4 mitogen-activated protein kinase kinase 4, MKK7 mitogen-activated protein kinase kinase 7, JNK c-Jun N-terminal kinase, GSK-3β glycogen synthase kinase 3β, Akt protein kinase B, PDK1 pyruvate dehydrogenase kinase 1, PTEN phosphatase and tensin homolog, c-Raf c-rapidly accelerated fibrosarcoma, TGF-β1 transforming growth factor-β1, IL-6 interleukin-6, TNF-α tumor necrosis factor-α

Article Snippet: The CM with 20 μg/ml transforming growth factor-β (TGF-β) neutralization antibody 1D11 (Thermo Fisher Scientific, USA, Cat# 16-9243-85; RRID: AB_2573124) or mouse IgG, was used to treat primary HSCs.

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Confocal Microscopy, Ligation, Knock-Out, Reverse Transcription Polymerase Chain Reaction

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Elevated TGFβ signaling contributes to ocular anterior segment dysgenesis in Col4a1 mutant mice

doi: 10.1016/j.matbio.2022.05.001

Figure Lengend Snippet: (A) Schematic illustration of the 1D11 administration paradigm for histological analysis. (B) Representative images of H&E stained sections (left) showing whole eyes (top panels) and corneas (lower panels) from E18.5 mice and quantification graph (right) showing increased corneal stromal thickness in Col4a1+/G1344D mice treated with 1D11 compared to those that received the control IgG1 antibody. Scale bars = 200 μm (top) and 50 μm (bottom). n = 20 and 24 corneas from IgG1- and 1D11-treated Col4a1+/+ mice, and 24 and 30 corneas from IgG1- and 1D11-treated Col4a1+/G1344D mice, respectively. (C) Schematic illustration of the 1D11 administration paradigm for qPCR analyses. (D) qPCR analyses revealed that 1D11 treatment partially prevented the increased expression of TGFβ target genes in anterior segments from P0 Col4a1+/G1344D mice compared to their IgG1-treated counterparts. n = 5–6 samples per genotype. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001, two-way ANOVA and Tukey’s multiple comparison test.

Article Snippet: Timed-pregnant B6 females crossed with Col4a1 +/ G1344D males were injected intraperitoneally with the 1D11 pan-TGFβ neutralizing antibody (clone 1D11.16.8, BioXCell, West Lebanon, NH) or IgG1 isotype control antibody (clone MOPC-21, BioXCell) diluted in inVivoPure Dilution Buffer (pH 7.0, BioXCell) (20 mg/kg) every other day from E8.5 to E16.5 and animals were harvested at E18.5 or P0 for histological and molecular analyses, respectively.

Techniques: Staining, Control, Expressing, Comparison

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Elevated TGFβ signaling contributes to ocular anterior segment dysgenesis in Col4a1 mutant mice

doi: 10.1016/j.matbio.2022.05.001

Figure Lengend Snippet: (A) Schematic illustration of the 1D11 administration paradigm for histological analysis. (B) Representative images of H&E stained sections (left) showing whole eyes (top panels) and corneas (lower panels) from E18.5 mice and quantification graph (right) showing increased corneal stromal thickness in Col4a1+/G1344D mice treated with 1D11 compared to those that received the control IgG1 antibody. Scale bars = 200 μm (top) and 50 μm (bottom). n = 20 and 24 corneas from IgG1- and 1D11-treated Col4a1+/+ mice, and 24 and 30 corneas from IgG1- and 1D11-treated Col4a1+/G1344D mice, respectively. (C) Schematic illustration of the 1D11 administration paradigm for qPCR analyses. (D) qPCR analyses revealed that 1D11 treatment partially prevented the increased expression of TGFβ target genes in anterior segments from P0 Col4a1+/G1344D mice compared to their IgG1-treated counterparts. n = 5–6 samples per genotype. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001, two-way ANOVA and Tukey’s multiple comparison test.

Article Snippet: 1D11 TGFβ neutralizing antibody treatment Timed-pregnant B6 females crossed with Col4a1 +/ G1344D males were injected intraperitoneally with the 1D11 pan-TGFβ neutralizing antibody (clone 1D11.16.8, BioXCell, West Lebanon, NH) or IgG1 isotype control antibody (clone MOPC-21, BioXCell) diluted in inVivoPure Dilution Buffer (pH 7.0, BioXCell) (20 mg/kg) every other day from E8.5 to E16.5 and animals were harvested at E18.5 or P0 for histological and molecular analyses, respectively.

Techniques: Staining, Control, Expressing, Comparison