Analysis of the Ubiquitination and Phosphorylation of Vangl Proteins.

The core planar cell polarity (PCP) protein Vang/Vangl, including Vangl1 and Vangl2 in vertebrates, is indispensable during development. Our previous studies showed that the activity of Vangl is tightly controlled by two important posttranslational modifications, ubiquitination and phosphorylation. Vangl is ubiquitinated through an endoplasmic reticulum-associated degradation (ERAD) pathway and is phosphorylated by casein kinase 1 (CK1) in response to Wnt. Here, we present step-by-step procedures to analyze Vangl ubiquitination and phosphorylation, including cell culture, transfection, sample preparation, and signal detection, as well as the use of newly available phospho-specific antibodies to detect Wnt-induced Vangl2 phosphorylation. The protocol described here can be applicable to the analysis of posttranslational modifications of other membrane proteins.