Wnt proteins are glycoproteins that have palmitoleate attached and are important in cell proliferation and cancer. In humans, they participate in several signaling pathways, which are divided into canonical and non-canonical signaling pathways. In the canonical signaling pathway, the transcriptional regulator β-catenin is important, to which, the intracellular part of the tumor marker EpCAM can bind. The extracellular part of EpCAM (EpEX) contains a hydrophobic pocket into which palmitate can bind. As EpCAM proteins and Wnt signaling pathway proteins are often mentioned in the literature in the same context, we wanted to express some selected proteins in HEK293T cells and check whether EpEX can bind them via its hydrophobic pocket. HEK293T cells were transfected under different conditions with plasmids containing transcripts for V5-tagged Wnt proteins. After transfection, we performed a pull-down assay with Wnt proteins and the unmutated or mutated (closed pocket) form of EpEX. Both forms were previously prepared in insect cells and purified by nickel affinity chromatography. The results after the transfections showed that Wnt proteins are expressed in small amounts in the cells. We did not detect any band on the membrane after the pull-down assay and the following Western blot. The reason for the absence of band could be a small amount of expressed Wnt proteins, due to which the signal cannot be detected. Another possibility is that the interaction between Wnt and EpEX is absent or weak. In further studies, it would therefore be necessary to optimize the transfection conditions and recheck the potential interaction between EpEX and Wnt.
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