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. 2020 Jun 12;9(6):125. doi: 10.3390/biology9060125

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© 2020 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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Protein-lipid overlay assay. In vitro protein-lipid overlay assay was carried out by incubating PIP-strips that were immobilized with 100 pmol of 15 lipids (A) with a purified PI-binding protein, 2× Hrs-1D4, FAPP1-1D4, TAPP1-1D4, PLCδ-1D4, and Grp1-1D4, followed by immunoblot analysis with the anti-rhodopsin antibody (A). The PI ELISA assay was carried out with different concentrations of 2× Hrs (0.25, 0.5, 1.0, and 2.0 µg/mL) using 2.5 pmols of PI(3)P, PI(4)P, PI(5)P, PI(3,4)P₂, PI(4,5)P₂, PI(3,4,5)P₃, and PC/PE/PS (15:35:15) (B). The PI assay was carried as described in the methods. Data are mean ± SEM (n = 3).