2. Academic Validation
  3. A pull-down and slot blot-based screening system for inhibitor compounds of the podoplanin-CLEC-2 interaction

A pull-down and slot blot-based screening system for inhibitor compounds of the podoplanin-CLEC-2 interaction

  • PLoS One. 2019 Sep 25;14(9):e0222331. doi: 10.1371/journal.pone.0222331.
Nobuo Watanabe 1,  Masako Kidokoro 1,  Yusuke Suzuki 1,  Makiko Tanaka 1,  Shigeaki Inoue 1,  Hideo Tsukamoto 2,  Noriaki Hirayama 3,  Pei-Wen Hsieh 4 5,  Ching-Ping Tseng 6 7 8,  Yoshihide Nakagawa 1,  Sadaki Inokuchi 1
Affiliations

Affiliations

  • 1 Department of Emergency and Critical Care Medicine, Tokai University School of Medicine, Isehara, Kanagawa, Japan.
  • 2 Department of the Education and the Research Support Center Tokai University School of Medicine, Isehara, Kanagawa, Japan.
  • 3 Institute of Advanced Biosciences, Tokai University, Isehara, Kanagawa, Japan.
  • 4 Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan, Taiwan, Republic of China.
  • 5 Department of Anesthesiology, Chang Gung Memorial Hospital, Taoyuan, Taiwan, Republic of China.
  • 6 Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, Taoyuan, Taiwan, Republic of China.
  • 7 Graduate Institute of Biomedical Science, College of Medicine, Chang Gung University, Taoyuan, Taiwan, Republic of China.
  • 8 Department of Laboratory Medicine, Chang Gung Memorial Hospital, Taoyuan, Taiwan, Republic of China.
PMID: 31553741 DOI: 10.1371/journal.pone.0222331
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.

Figures
Products
嗨！很高兴为您提供帮助！

尊敬的 MCE 客户您好，
请您选择所在区域，我们将转接对应客服为您服务！

热门区域

A

B

C

F

G

H

J

L

N

Q

S

T

X

Y

Z

热 A B C F G H J L N Q S T X Y Z