Antibody microarray and immunoblotting analyses of the EGF signaling phosphorylation network in human A431 epidermoid carcinoma cells

Abstract

Post-homogenization instability of phosphorylation sites in proteins, and the insensitivity and high costs of most proteomics analytical methods have been major barriers in tracking cell signalling networks in minute specimens of cells and tissues. While antibody microarrays have been promising tools for quantifying changes in protein expression and phosphorylation, the interpretation of findings from their applications have been complicated by issues of protein-protein interactions and cross-reactivity with off-target proteins. We used the human A431 cervical carcinoma cell line that over-expresses the epidermal growth factor (EGF) receptor to investigate its signal transduction mechanisms. Chemical cleavage of proteins at cysteine residues at the time of homogenizing the A431 cells permitted preservation of the autophosphorylation of this receptor in response to brief stimulation of these cells with EGF and revealed many downstream signalling events. Most of the EGF-induced changes in protein phosphorylation observed with the antibody microarray were validated by Western blotting with the intended targets, although many of these also false positives that arose from cross-reactivity with the EGF receptor itself. Some false-negatives from blocked epitopes may have stemmed from interactions with SH2 domain-containing adapter proteins that were resistant to chemical cleavage and internal interactions of flanking arginine and lysine residues with phosphosites.

FAK-Mediated Signaling Controls Amyloid Beta Overload, Learning and Memory Deficits in a Mouse Model of Alzheimer’s Disease

“…Hippocampal punches were collected from control and Ptk2 -OE injected mice at the end of behavioral tests and subjected to Kinexus Kinex TM KAM-1325 antibody microarray analyses as described [ 74 ]. These microarrays feature about 1350 commercial antibodies produced principally by Kinexus as well as from other suppliers following their in-house validation, with each antibody printed in quadruplicate on each Nexterion P slide (Schott AG, Jena, Germany).…”

CAT PETR: A Graphical User Interface for Differential Analysis of Phosphorylation and Expression Data

“…Antibody microarrays use immobilized antibody spots to capture specific targets from a given sample, allowing for the binding of many different targets to be quantified [1]. The ever growing number of array validated antibodies have made antibody microarrays an increasingly powerful tool for biomarker and drug target discovery [2], protein modification analysis [3][4][5], and proteinprotein interaction detection [6]. Yet, computational tools are still lacking to efficiently analyze the data.…”