Characterization of a rabbit polyclonal antibody against threonine-AMPylation

Abstract

An antibody against the posttranslational modification AMPylation was produced using a peptide corresponding to human Rac1 switch I region with AMPylated threonine-35 residue as an antigen. The resulting rabbit antiserum was tested for its abilities to recognize AMPylated proteins by western blot and immunoprecipitation. The antiserum is highly specific for threonine-AMPylated proteins and weakly recognizes tyrosine-AMPylated proteins. Depletion of serum with modified protein abolished its activity against tyrosine-AMPylated proteins. The antiserum also recognized native proteins with modification in an immunoprecipitation experiment. Interactions of the antiserum could be inhibited by competition with AMP but not with GMP or UMP. This antiserum had potential utility for the identification of unknown AMPylated proteins.

Unfolded Protein Response-regulated Drosophila Fic (dFic) Protein Reversibly AMPylates BiP Chaperone during Endoplasmic Reticulum Homeostasis

“…Samples were analyzed by autoradiography after SDS-PAGE. For the assays where AMPylation was detected by the anti-AMP-Thr antibody (42), the experiment was performed as described above but excluding radiolabeled ATP.…”

“…We treated S2 cells with tunicamycin, a pharmacological inducer of ER stress, for different times and blotted the whole cell lysate with anti-AMP-Thr antibody (42). A protein with the size of BiP was AMPylated in S2 cells, but AMPylation started to decrease within 30 min of tunicamycin treatment and was completely absent after 24 h (Fig.…”

HypE-specific Nanobodies as Tools to Modulate HypE-mediated Target AMPylation

“…A threonine/AMP-specific mouse serum was used to confirm VopS-mediated AMPylation of Cdc42 and Rac1 and to study HypE-mediated BiP AMPylation (21). In addition, a set of small molecules inhibiting VopS in in vitro assays has been characterized (22).…”

Global Profiling of Huntingtin-associated protein E (HYPE)-Mediated AMPylation through a Chemical Proteomic Approach

“…AMPylation profiling is not a trivial task (19), and several strategies have emerged over the past few years ranging from labeling with radioactive ATP (2, 3) and immunoprecipitation with AMPylation-specific antibodies (20,21) to mass spectrometry (MS) approaches focused on AMP fragmentation (22,23). Although these methods contributed significantly to developments in the field, they also suffer from certain drawbacks, including low sensitivity, high background, limited quantitative power, and limited amenability to high-throughput (HT) substrate identification.…”