Co-immunoprecipitation Assays

Evans, Ian M.; Paliashvili, Ketevan

doi:10.1007/978-1-0716-2217-9_8

Cited by 13 publications

(9 citation statements)

References 3 publications

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“…The different bands were collected for further LC‒MS/MS analysis of USP7-interacting proteins in PBMCs. As mentioned above, Western blotting was performed to confirm the interaction between the two proteins [ 23 , 24 ].…”

Section: Methodsmentioning

confidence: 99%

USP7 promotes the osteoclast differentiation of CD14+ human peripheral blood monocytes in osteoporosis via HMGB1 deubiquitination

Lin¹,

Zheng²,

Liu³

et al. 2023

Journal of Orthopaedic Translation

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Section: Methodsmentioning

confidence: 99%

USP7 promotes the osteoclast differentiation of CD14+ human peripheral blood monocytes in osteoporosis via HMGB1 deubiquitination

Lin¹,

Zheng²,

Liu³

et al. 2023

Journal of Orthopaedic Translation

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“…Coimmunoprecipitation (co‐IP) was performed according to a previously published approach. [ 26 ] Briefly, HEK293T cells were transfected with Flag‐tagged sdAbs and HA‐tagged GSDME constructs. After 48 h, cells were collected and sonicated in lysis buffer (50 mM Tris‐HCl pH 7.4, 150 mM NaCl, 1% IGEPAL CA‐630, 0.2 mM EDTA, pH 8.0) followed by centrifugation at 14,000 × g for 10 min.…”

Section: Methodsmentioning

confidence: 99%

Engineering single‐domain antibodies targeting Gasdermin E activation by the chemotherapeutic agent cis‐diaminodichloroplatinum

Gao

et al. 2023

Biotechnology Journal

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As mediators of pyroptosis, gasdermins (GSDMs) are closely associated with systemic cytotoxicity or so‐called side effects and are also involved in the inflammatory response during chemotherapy. Using in situ proximity ligation assay followed by sequencing (isPLA‐seq), which we recently developed, we screened a single‐domain antibody (sdAb) library and identified several sdAbs against Gasdermin E (GSDME) that specifically recognize the N‐terminal domain (1‐270 aa) of GSDME (GSDME‐NT). One of them mitigated the release of inflammatory damage‐associated molecular patterns (DAMPs) and cytokines, including high mobility group protein b1 (Hmgb1) and interleukin‐1β (Il‐1β), in isolated mouse alveolar epithelial cells (AECs) upon chemotherapeutic agent cis‐diaminodichloroplatinum (CDDP) treatment. Further investigation showed that this anti‐GSDME sdAb also alleviated CDDP‐induced pyroptotic cell death and lung tissue injury and decreased systemic Hmgb1 release in C57/BL6 mice, due to GSDME inactivation. Collectively, our data define an inhibitory role of the specific sdAb against GSDME, providing a potential strategy for systemically alleviating chemotherapeutic toxicities in vivo.

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“…The assay was performed as previously described. [ 40 ] In brief, after lysis in IP lysis buffer as well as sonication on ice, the cell lysate was precipitated by centrifugation. For IP, cell lysate was treated with β‐arrestin‐1 or H4K16ac antibody with rotation followed by treatment with protein A/G‐conjugated agarose beads.…”

Section: Methodsmentioning

confidence: 99%

T‐2 Toxin‐Mediated β‐Arrestin‐1 O‐GlcNAcylation Exacerbates Glomerular Podocyte Injury via Regulating Histone Acetylation

Li,

Sun,

Zhu

et al. 2023

Advanced Science

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T‐2 toxin causes renal dysfunction with proteinuria and glomerular podocyte damage. This work explores the role of metabolic disorder/reprogramming‐mediated epigenetic modification in the progression of T‐2 toxin‐stimulated podocyte injury. A metabolomics experiment is performed to assess metabolic responses to T‐2 toxin infection in human podocytes. Roles of protein O‐linked‐N‐acetylglucosaminylation (O‐GlcNAcylation) in regulating T‐2 toxin‐stimulated podocyte injury in mouse and podocyte models are assessed. O‐GlcNAc target proteins are recognized by mass spectrometry and co‐immunoprecipitation experiments. Moreover, histone acetylation and autophagy levels are measured. T‐2 toxin infection upregulates glucose transporter type 1 (GLUT1) expression and enhances hexosamine biosynthetic pathway in glomerular podocytes, resulting in a significant increase in β‐arrestin‐1 O‐GlcNAcylation. Decreasing β‐arrestin‐1 or O‐GlcNAc transferase (OGT) effectively prevents T‐2 toxin‐induced renal dysfunction and podocyte injury. Mechanistically, O‐GlcNAcylation of β‐arrestin‐1 stabilizes β‐arrestin‐1 to activate the mammalian target of rapamycin (mTOR) pathway as well as to inhibit autophagy during podocyte injury by promoting H4K16 acetylation. To sum up, OGT‐mediated β‐arrestin‐1 O‐GlcNAcylation is a vital regulator in the development of T‐2 toxin‐stimulated podocyte injury via activating the mTOR pathway to suppress autophagy. Targeting β‐arrestin‐1 or OGT can be a potential therapy for T‐2 toxin infection‐associated glomerular injury, especially podocyte injury.

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Co-immunoprecipitation Assays

Cited by 13 publications

References 3 publications

USP7 promotes the osteoclast differentiation of CD14+ human peripheral blood monocytes in osteoporosis via HMGB1 deubiquitination

USP7 promotes the osteoclast differentiation of CD14+ human peripheral blood monocytes in osteoporosis via HMGB1 deubiquitination

Engineering single‐domain antibodies targeting Gasdermin E activation by the chemotherapeutic agent cis‐diaminodichloroplatinum

T‐2 Toxin‐Mediated β‐Arrestin‐1 O‐GlcNAcylation Exacerbates Glomerular Podocyte Injury via Regulating Histone Acetylation

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