Author/Authors :
Erbilgin, Yücel Istanbul Üniversitesi - Deneysel Tip Arastirma Enstitüsü - Genetik Anabilim Dali, Turkey , Ng, Özden Hatirnaz Istanbul Üniversitesi - Deneysel Tip Arastirma Enstitüsü - Genetik Anabilim Dali, Turkey , Sayitoglu, Müge Istanbul Üniversitesi - Deneysel Tip Arastirma Enstitüsü - Genetik Anabilim Dali, Turkey , Söderberg, Ola Uppsala University - Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Sweden , Özbek, Ugur Istanbul Üniversitesi - Deneysel Tip Arastirma Enstitüsü - Genetik Anabilim Dali, Turkey
Abstract :
Objective: Analyses of protein-protein interactions (PPIs) are attracting more and more attention in the field of proteomics, caused by the necessity of in-depth functional study of proteins and signaling networks. Proximity ligation assay (PLA) is a novel method that has the advantage that it can be utilized for detection of endogenous proteins and PPIs in cultured cells and tissue sections. The aim of the study was to determine active B-catenin and Axin2 interactions, which are members of the WNT pathway, in the T-cell acute lymphoblastic leukemia (T-ALL) cell line- Jurkat using in situ PLA. Materials and Methods: Jurkat cells were cultured in RPMI media and fixed on glass slides. After permeabilization, the slides were blocked and incubated with primary antibodies and PLA probes. Hybridization and ligation of the circularization oligonucleotides were then performed to generate a substrate for the subsequent rolling circle amplification (RCA). The RCA products were visulaized by hybridization of Texas Red labelled detection probes and the resulting in situ PLA signals were then detected by fluorescence microscopy. Results: Both B-catenin and Axin2 proteins were found to be expressed in Jurkat cells and interactions between these proteins were detected both in nucleus and cytoplasm. Conclusion: The resulted presented herein show that in situ PLA could be used to visualize interactions between endogenously expressed WNT pathway members in single cells. We detected active B-catenin-Axin2 interaction in the T-ALL cell line Jurkat, a sign of deregulated WNT signaling which could be a potantial target for future therapeutics.
