Generation of Pax1/PAX1-Specific Monoclonal Antibodies

Feederle, Regina; Gerber, Josef-Karl; Middleton, Amy; Northrup, Emily; Kist, Ralf; Kremmer, Elisabeth; Peters, Heiko

doi:10.1089/mab.2016.0029

Cited by 10 publications

(8 citation statements)

References 18 publications

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“…To generate an antibody against murine Regnase-3 protein, aa 1–903 of the Regnase-3 protein sequence with N-terminal GST-tag and C-terminal His-tag were expressed in Escherichia coli and purified in two steps on a HiTrap Chelating and a GSTrap column. Lou/C rats and C57BL/6 mice were immunized with the Regnase-3 peptide as described elsewhere (Feederle et al, 2016). Specific antibodies were first selected by enzyme-linked immunoassay on the protein and, in a second step, by immunoblotting using GFP-Regnase-3 protein lysates, expressed in HEK293T cells.…”

Section: Methodsmentioning

confidence: 99%

Immune homeostasis and regulation of the interferon pathway require myeloid-derived Regnase-3

Gamm

Schaub

Jones

et al. 2019

Journal of Experimental Medicine

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The RNase Regnase-1 is a master RNA regulator in macrophages and T cells that degrades cellular and viral RNA upon NF-κB signaling. The roles of its family members, however, remain largely unknown. Here, we analyzed Regnase-3–deficient mice, which develop hypertrophic lymph nodes. We used various mice with immune cell–specific deletions of Regnase-3 to demonstrate that Regnase-3 acts specifically within myeloid cells. Regnase-3 deficiency systemically increased IFN signaling, which increased the proportion of immature B and innate immune cells, and suppressed follicle and germinal center formation. Expression analysis revealed that Regnase-3 and Regnase-1 share protein degradation pathways. Unlike Regnase-1, Regnase-3 expression is high specifically in macrophages and is transcriptionally controlled by IFN signaling. Although direct targets in macrophages remain unknown, Regnase-3 can bind, degrade, and regulate mRNAs, such as Zc3h12a (Regnase-1), in vitro. These data indicate that Regnase-3, like Regnase-1, is an RNase essential for immune homeostasis but has diverged as key regulator in the IFN pathway in macrophages.

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Section: Methodsmentioning

confidence: 99%

Immune homeostasis and regulation of the interferon pathway require myeloid-derived Regnase-3

Gamm

Schaub

Jones

et al. 2019

Journal of Experimental Medicine

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“…To generate antibodies against human YKL-39, Lou/C rats and C57 BL/6 mice were immunized with ovalbumin-coupled peptides spanning aa 364–377 and 133–154, respectively, using standard procedures as described. 79 Specificity of antibodies was verified by enzyme-linked immunoassay on peptides and by Western blotting using stably transfected HEK293-YKL-39-FLAG and HEK293-vector cells generated by stable transfection with plasmids pSNAP-YKL-39-FLAG and pSNAP-tag. The hybridoma cells of YKL-39-reactive supernatants were cloned at least twice by limiting dilution.…”

Section: Methodsmentioning

confidence: 99%

Tumor-associated macrophages in human breast cancer produce new monocyte attracting and pro-angiogenic factor YKL-39 indicative for increased metastasis after neoadjuvant chemotherapy

et al. 2018

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In breast cancer, the tumor microenvironment plays a critical role in the tumor progression and responses to therapy. Tumor-associated macrophages (TAMs) are major innate immune cells in tumor microenvironment that regulate intratumoral immunity and angiogenesis by secretion of cytokines, growth factors as well as chitinase-like proteins (CLPs), that combine properties of cytokines and growth factors. YKL-39 is a chitinase-like protein found in human and absent in rodents, and its expression in TAMs and role in breast cancer progression was not studied to date. Here for the first time we demonstrate that YKL-39 is expressed on TAMs, predominantly positive for stabilin-1, but not by malignant cells or other stromal cells in human breast cancer. TGF-beta in combination with IL-4, but not IL-4 alone was responsible of the stimulation of the production of YKL-39 in human primary macrophages. Mechanistically, stabilin-1 directly interacted with YKL-39 and acted as sorting receptor for targeting YKL-39 into the secretory pathway. Functionally, purified YKL-39 acted as a strong chemotactic factor for primary human monocytes, and induced angiogenesis in vitro. Elevated levels of YKL-39 expression in tumors after neoadjuvant chemotherapy (NAC) were predictive for increased risk of distant metastasis and for poor response to NAC in patients with nonspecific invasive breast carcinoma. Our findings suggest YKL-39 as a novel therapeutic target, and blocking of its activity can be combined with NAC in order to reduce the risk of metastasis in breast cancer patients.

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“…Expression and purification of an N-terminal fragment of ASXL1 (amino acids 1–618), followed by immunization of Lou/c rats was performed as previously described (Feederle et al., 2016, Studier, 2005).…”

Section: Methodsmentioning

confidence: 99%

Pathological ASXL1 Mutations and Protein Variants Impair Neural Crest Development

et al. 2019

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Summary The neural crest (NC) gives rise to a multitude of fetal tissues, and its misregulation is implicated in congenital malformations. Here, we investigated molecular mechanisms pertaining to NC-related symptoms in Bohring-Opitz syndrome (BOS), a developmental disorder linked to mutations in the Polycomb group factor Additional sex combs-like 1 ( ASXL1 ). Genetically edited human pluripotent stem cell lines that were differentiated to NC progenitors and then xenotransplanted into chicken embryos demonstrated an impairment of NC delamination and emigration. Molecular analysis showed that ASXL1 mutations correlated with reduced activation of the transcription factor ZIC1 and the NC gene regulatory network. These findings were supported by differentiation experiments using BOS patient-derived induced pluripotent stem cell lines. Expression of truncated ASXL1 isoforms (amino acids 1–900) recapitulated the NC phenotypes in vitro and in ovo , raising the possibility that truncated ASXL1 variants contribute to BOS pathology. Collectively, we expand the understanding of truncated ASXL1 in BOS and in the human NC.

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Generation of Pax1/PAX1-Specific Monoclonal Antibodies

Cited by 10 publications

References 18 publications

Immune homeostasis and regulation of the interferon pathway require myeloid-derived Regnase-3

Immune homeostasis and regulation of the interferon pathway require myeloid-derived Regnase-3

Tumor-associated macrophages in human breast cancer produce new monocyte attracting and pro-angiogenic factor YKL-39 indicative for increased metastasis after neoadjuvant chemotherapy

Pathological ASXL1 Mutations and Protein Variants Impair Neural Crest Development

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