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Updated: Aug 24, 2026
Isolation of Whole Cell Protein Lysates from Mouse Facial Processes and Cultured Palatal Mesenchyme Cells for Phosphoprotein Analysis
Published on: April 1, 2022
Phosphopeptide mapping of proteins ectopically expressed in tissue culture cell lines
Beth A. Firulli1, David M. Virshup, Anthony B. Firulli
1Wells Center for Pediatric Research, James Whitcomb Riley Hospital for Children, Departments of Pediatrics and Medical & Molecular Genetics. Indiana University Medical School, 1044 W. Walnut, R4 402 Indianapolis, IN 46202-5225. USA.
Abstract:
Post-translational modifications such as phosphorylation play a vital role in the regulation of protein function. In our study of the basic Helix-loop-Helix (bHLH) transcription factor HAND1, it was suspected that HAND1 was being phosphorylated during trophoblast giant cell differentiation and that coexpression of a constitutively active kinase with HAND1 resulted in changes in the proteins dimerization profile. In order to accurately document HAND1 phosphorylation and identify the resides being modified, we employed metabolic cell labeling with (32)P of tissue culture cells coexpressing a Flag-epitope tagged HAND1 along with a number of active kinases and phosphatase subunits. We generated phosphopeptide maps of the phosphorylated HAND1 using the methods described below and linked these modifications to changes in HAND1 biological function.
