Plant antigens cross‐react with rat polyclonal antibodies against KLH‐conjugated peptides

Oulehlová, Denisa; Hála, Michal; Potocký, Martin; Žárský, Viktor; Cvrčková, Fatima

doi:10.1016/j.cellbi.2008.10.003

Cited by 4 publications

(2 citation statements)

References 19 publications

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“…3E,F). A similar problem associated with the non-specific binding the aforementioned IFS antibody has already been described by Jaganath (2005) and can be ascribed to the fact that polyclonal antibodies raised against the KLH:peptide conjugates can cross-react with various plant antigens in Western blot analysis, thus causing the non-specificity of the immunodetection (Oulehlová et al 2009).…”

Section: Resultsmentioning

confidence: 61%

Functional expression and subcellular localization of pea polymorphic isoflavone synthase CYP93C18

Pičmanová¹,

Reňák²,

Feciková³

et al. 2013

Biologia plant.

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Isoflavone synthase (IFS; CYP93C) plays a key role in the biosynthesis of phenolic secondary metabolites, isoflavonoids. These compounds, which are well-known for their benefits to human health and plant defence, are produced mostly in legumes. However, more than 200 of them have been described in 59 other plant families without any knowledge of their respective IFS orthologue genes (with the sole exception of sugar beet). In this study, we selected IFS from Pisum sativum L. (CYP93C18) for functional expression. CYP93C18 was isolated, cloned, and introduced into Arabidopsis thaliana. The presence of the gene was shown by Southern blot analysis and its expression in the transgenic Arabidopsis was proven by RT-PCR and Western blots. The functional activity of the heterologous IFS was verified by HPLC-MS analysis of the metabolite levels: the isoflavone genistein and its derivatives tectorigenin and biochanin A were detected in the overexpressing lines. In addition, 35S::CYP93C18::GFP fused proteins were transiently expressed in the leaves of Nicotiana benthamiana and the localization of the GFP signal was observed on the endoplasmic reticulum using confocal microscopy which is consistent with the data from the literature and with our in silico predictions. The putative mode of attachment of IFS to the endoplasmic reticulum membrane is suggested. The undemanding methodology presented in this paper is applicable to the functional analysis of newly-identified isoflavone synthase genes from various species.

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Section: Resultsmentioning

confidence: 61%

Functional expression and subcellular localization of pea polymorphic isoflavone synthase CYP93C18

Pičmanová¹,

Reňák²,

Feciková³

et al. 2013

Biologia plant.

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“…Although protein tagging by fusion with a relatively large polypeptide such as the YFP or GFP might influence its intracellular localization, such fusion proteins, including a plant formin [35], have been documented to be functional. Unfortunately, we could not confirm our observations by an independent method since we were unable to obtain specific antibodies suitable for immunostaining of formins, including AtFH13 [36]. Another theoretical alternative, expression of small tags visualized using cell permeable ligands, is well established in mammalian cell biology but practically restricted to the study of extracytoplasmic proteins in plants [37], with the exception of a single report with no published experimental follow-up [38].…”

Section: Discussionmentioning

confidence: 81%

Arabidopsis Class II Formins AtFH13 and AtFH14 Can Form Heterodimers but Exhibit Distinct Patterns of Cellular Localization

Kollárová

Forero

Stillerová

et al. 2020

IJMS

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Formins are evolutionarily conserved multi-domain proteins participating in the control of both actin and microtubule dynamics. Angiosperm formins form two evolutionarily distinct families, Class I and Class II, with class-specific domain layouts. The model plant Arabidopsis thaliana has 21 formin-encoding loci, including 10 Class II members. In this study, we analyze the subcellular localization of two A. thaliana Class II formins exhibiting typical domain organization, the so far uncharacterized formin AtFH13 (At5g58160) and its distant homolog AtFH14 (At1g31810), previously reported to bind microtubules. Fluorescent protein-tagged full length formins and their individual domains were transiently expressed in Nicotiana benthamiana leaves under the control of a constitutive promoter and their subcellular localization (including co-localization with cytoskeletal structures and the endoplasmic reticulum) was examined using confocal microscopy. While the two formins exhibit distinct and only partially overlapping localization patterns, they both associate with microtubules via the conserved formin homology 2 (FH2) domain and with the periphery of the endoplasmic reticulum, at least in part via the N-terminal PTEN (Phosphatase and Tensin)-like domain. Surprisingly, FH2 domains of AtFH13 and AtFH14 can form heterodimers in the yeast two-hybrid assay—a first case of potentially biologically relevant formin heterodimerization mediated solely by the FH2 domain.

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Native Chromatin Immunoprecipitation

Cosseau

Grunau

2011

Methods in Molecular Biology

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Native chromatin immunoprecipitation refers to a method that allows for identification and quantification of DNA that is associated with specific chromatin proteins without altering the structure of these proteins. The method has been used with great success in the past and has some advantages over the more widely used cross-linking chromatin immunoprecipitation. We describe here a protocol that was specifically optimized for low cell numbers.

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Plant antigens cross‐react with rat polyclonal antibodies against KLH‐conjugated peptides

Cited by 4 publications

References 19 publications

Functional expression and subcellular localization of pea polymorphic isoflavone synthase CYP93C18

Functional expression and subcellular localization of pea polymorphic isoflavone synthase CYP93C18

Arabidopsis Class II Formins AtFH13 and AtFH14 Can Form Heterodimers but Exhibit Distinct Patterns of Cellular Localization

Native Chromatin Immunoprecipitation

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