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Development and characterization of a novel monoclonal antibody that recognizes an epitope in the central protein interaction domain of RapGEF1 (C3G)

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Abstract

The ubiquitously expressed protein RapGEF1 (C3G) regulates multiple cellular activities and is essential for early embryonic development in mammals. It has functions dependent on its catalytic activity as well as protein interaction domain and regulates β-catenin signaling. This study describes the generation of a novel monoclonal antibody, 3F6mAb and its characterization for recognition of RapGEF1. Mice were immunized with recombinant protein having only the Crk binding region of RapGEF1 and hybridoma clones created by fusion of immunized spleen cells with Sp2/0 myeloma cells. This antibody recognizes human, primate and murine RapGEF1 protein. Based on the recognition of various deletion constructs, we have mapped its epitope to 580–648 amino acids. Isotyping showed that it belongs to IgG1 class of heavy chain and Kappa light chain. 3F6mAb is suitable for detecting cellular RapGEF1 by western-blotting, immunofluorescence and immunoprecipitation. It has an advantage over most of the commercially available antibodies as it can detect N- and C-terminal truncated forms of RapGEF1. Using this antibody to detect mobility shift, we show that RapGEF1 is phosphorylated on tyrosine as well as S/T residues in its Crk binding domain. This monoclonal antibody is a valuable tool that will aid in understanding functions of cellular RapGEF1.

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Abbreviations

RapGEF1:

Rap guanine nucleotide exchange factor 1

CBR:

Crk-binding region

SFK:

Src family kinases

HAT:

Hypoxanthine-aminopterin-thymidine

GSK3β:

Glycogen synthase kinase beta

PV:

Pervanadate

OA:

Okadaic acid

LiCl:

Lithium chloride

TCPTP:

T-cell protein tyrosine phosphatase

ELISA:

Enzyme-linked immunosorbent assay

GST:

Glutathione-S-transferase

GFP:

Green fluorescent protein

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Acknowledgements

We thank S. Tanaka (The Rockefeller University, New York) and P. J. S. Stork (Oregon Health and Science University), for gifts of valuable plasmids. We thank AK Raghawan for help with making figures. D.S acknowledges a fellowship from the Council of Scientific and Industrial Research, Government of India. This work was supported by funds from the Department of Biotechnology, Government of India (BT/PR11759/BRB/10/1301/2014) and CSIR (BSC0111), awarded to VR.

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  1. Zareena Begum and Ch. Varalakshmi have contributed equally.

Authors and Affiliations

  1. Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad, 500 007, India

    Zareena Begum, Ch. Varalakshmi, Divya Sriram & Vegesna Radha

Authors
  1. Zareena Begum
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  2. Ch. Varalakshmi
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  3. Divya Sriram
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  4. Vegesna Radha
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Correspondence to Vegesna Radha.

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Ethical approval

Institutional Animal Ethics Committee clearance was obtained for use of animals in the study (IAEC no. 45/2013). All animal experiments and procedures were performed in accordance with the relevant guidelines and regulations on ethical standards for investigation of animals framed by The Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Govt. of India.

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Begum, Z., Varalakshmi, C., Sriram, D. et al. Development and characterization of a novel monoclonal antibody that recognizes an epitope in the central protein interaction domain of RapGEF1 (C3G). Mol Biol Rep 45, 1809–1819 (2018). https://doi.org/10.1007/s11033-018-4327-0

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