Detection of SUN1 Splicing Variants at the mRNA and Protein Levels in Cancer

Abstract

The linker of nucleoskeleton and cytoskeleton (LINC) complex, containing the proteins SUN and nesprin, is the fundamental structural unit of the nuclear envelope. The neoplastic-based regulation of the LINC complex in cancer tissues has become increasingly recognized in recent years, including the altered expression, somatic mutation, and methylation of genes. However, precisely how mutations and deregulated expression of the LINC complex contribute to the pathogenic mechanisms of tumorigenesis remain to be elucidated, mainly because of several technical difficulties. First, both the SUN and SYNE (encoding nesprin) genes give rise to a vast number of splicing variants. Second, immunoprecipitation experiments of endogenous SUN and nesprin proteins are difficult owing to the lack of suitable reagents as well as the limited solubility of these proteins in mild extraction conditions. Here, we describe three protocols to investigate these aspects: (1) immunohistochemistry to determine the expression levels and localization of the LINC complex in cancer tissue, (2) detection of SUN1 splicing variants at the mRNA level, and (3) detection of SUN1 splicing variants and binding partners at the protein level.