Unknown

Dataset Information

0

Detection and Quantification of ADP-Ribosylated RhoA/B by Monoclonal Antibody.


ABSTRACT: Clostridium botulinum exoenzyme C3 is the prototype of C3-like ADP-ribosyltransferases that modify the GTPases RhoA, B, and C. C3 catalyzes the transfer of an ADP-ribose moiety from the co-substrate nicotinamide adenine dinucleotide (NAD) to asparagine-41 of Rho-GTPases. Although C3 does not possess cell-binding/-translocation domains, C3 is able to efficiently enter intact cells, including neuronal and macrophage-like cells. Conventionally, the detection of C3 uptake into cells is carried out via the gel-shift assay of modified RhoA. Since this gel-shift assay does not always provide clear, evaluable results an additional method to confirm the ADP-ribosylation of RhoA is necessary. Therefore, a new monoclonal antibody has been generated that specifically detects ADP-ribosylated RhoA/B, but not RhoC, in Western blot and immunohistochemical assay. The scFv antibody fragment was selected by phage display using the human naive antibody gene libraries HAL9/10. Subsequently, the antibody was produced as scFv-Fc and was found to be as sensitive as a commercially available RhoA antibody providing reproducible and specific results. We demonstrate that this specific antibody can be successfully applied for the analysis of ADP-ribosylated RhoA/B in C3-treated Chinese hamster ovary (CHO) and HT22 cells. Moreover, ADP-ribosylation of RhoA was detected within 10 min in C3-treated CHO wild-type cells, indicative of C3 cell entry.

SUBMITTER: Rohrbeck A 

PROVIDER: S-EPMC4848626 | BioStudies | 2016-01-01

REPOSITORIES: biostudies

Similar Datasets

1995-01-01 | S-EPMC1136802 | BioStudies
2005-01-01 | S-EPMC556266 | BioStudies
1994-01-01 | S-EPMC1138134 | BioStudies
2015-01-01 | S-EPMC4528107 | BioStudies
2017-01-01 | S-EPMC5310985 | BioStudies
2019-01-01 | S-EPMC6456868 | BioStudies
2019-01-01 | S-EPMC7825248 | BioStudies
1997-01-01 | S-EPMC1218292 | BioStudies
2014-01-01 | S-EPMC4072758 | BioStudies
2005-01-01 | S-EPMC1276701 | BioStudies