RRC ID 57724
Author Watanabe N, Kidokoro M, Suzuki Y, Tanaka M, Inoue S, Tsukamoto H, Hirayama N, Hsieh PW, Tseng CP, Nakagawa Y, Inokuchi S.
Title A pull-down and slot blot-based screening system for inhibitor compounds of the podoplanin-CLEC-2 interaction.
Journal PLoS One
Abstract Podoplanin, a transmembrane glycoprotein, is overexpressed in certain types of tumors and induces platelet aggregation by binding to C-type lectin-like receptor 2 (CLEC-2) on the platelet membrane. Activated platelets release granule components, which in turn, trigger epithelial-mesenchymal transition and confer invasive capacity to the tumor cells. Therefore, blocking the podoplanin-CLEC-2 interaction by a small-molecule compound is a potential therapeutic strategy to prevent cancer metastasis and invasion. To effectively identify such inhibitory compounds, we have developed a pull-down-based inhibitory compound screening system. An immunoglobulin Fc domain-CLEC-2 fusion protein was used as a bait to capture podoplanin derived from podoplanin-overexpressing HeLa cells in the presence and absence of the test compound. The protein complex was then pulled down using protein A beads. To shorten the turnaround time, increase throughput, and decrease the workload for the operators, centrifugal filter units were employed to separate free and bound podoplanin, instead of using customary aspiration-centrifugation washing cycles. Slot blotting was also utilized in lieu of gel electrophoresis and electrical transfer. Thus, the use of our pull down screening system could facilitate the effective selection of potential inhibitor compounds of the podoplanin-CLEC-2 interaction for cancer therapy. Importantly, our methodology is also applicable to targeting other protein-protein interactions.
Volume 14(9)
Pages e0222331
Published 2019-9-25
DOI 10.1371/journal.pone.0222331
PII PONE-D-19-03552
PMID 31553741
PMC PMC6760769
MeSH Drug Evaluation, Preclinical / methods* HeLa Cells Humans Immunoglobulin Fc Fragments / metabolism Lectins, C-Type / antagonists & inhibitors* Lectins, C-Type / metabolism Membrane Glycoproteins / antagonists & inhibitors* Membrane Glycoproteins / metabolism Protein Binding Recombinant Proteins
IF 2.74
Times Cited 0
DNA material CSII-CMV-MCS-IRES2-Venus (RDB04383) pCMV-VSV-G-RSV-Rev (RDB04393) pCAG-HIVgp (RDB04394)