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. 2019 Feb 1;10:68. doi: 10.3389/fimmu.2019.00068

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Copyright © 2019 Pandey, Bakay, Hain, Strenkowski, Yermakova, Kushner, Orange and Hakonarson.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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CLEC16A interacts with the class C Vps-HOPS complex, modulates NK cell surface markers expression and depicts cytosolic localization. (A) Representative histogram (left) and mean fluorescent intensities (MFI) of cell surface receptor CD28, CD226, and NKp46 in Clec16a overexpressing cells and YTS-GFP control (n = 3 repeats) by flow cytometry analysis. (B) Reversal of receptor expression in siRNA mediated CLEC16A knockdown. Representative histogram (left) depicts percentage decrease in expression of CD28, CD226, and NKp46 in control and CLEC16A siRNA nucleofected cells. MFI graph (right) depicts increase in expression of CD28, CD226, and NKp46 in YTS-CLEC16A overexpressing cells and YTS-GFP control (n = 3 repeats). ^*P < 0.05, ^***P < 0.001 (unpaired two-tailed Student's t-test). (C) CLEC16A IP-pull down and representative immunoblot analysis of CLEC16A, Vps16A, Hrs, Actinin-4, and CD226 and Nrdp1. Membranes were stripped and re-probed for β-actin as a loading control (bottom). (D) Representative reverse IP-pull down and immunoblot of VPS16A and CLEC16A in YTS and YTS-CLEC16A. (E) Representative reverse IP-pull down and immunoblot of Hrs and CLEC16A in YTS and YTS-CLEC16A. (F) Representative reverse IP-pull down and immunoblot of Hrs and CLEC16A in YTS and YTS-CLEC16A. (G) Representative CLEC16A IP-pull down and immunoblot of CLEC16A, Ub, UBE1a, and ATG5 in YTS and YTS-CLEC16A. (H) Representative CLEC16A IP-pull down and immunoblot of CLEC16A, Ub, UBE1a, and ATG5 in YTS and YTS-CLEC16A cells pretreated with proteasomal inhibitor MG132. Membranes were stripped and re-probed for β-actin as a loading control for all pull down experiments (bottom). (I) Representative immunoblot depicting cytosolic localization of CLEC16A in YTS-CLEC16A and ex vivo NK cell fractioned lysates. Membranes were stripped and probed for GAPDH as loading control for cytosolic fraction, Rab5 (early endosomes marker), Rab7 (late endosomes marker), Tim-23 (mitochondrial membrane fraction), Histone H3 (Nucleus), ERp72 (endoplasmic reticulum (ER), Syntaxin 6 (Golgi), and Cytokeratin 18 (cytoskeleton loading control).