Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2018 May 7;217(5):1797–1813. doi: 10.1083/jcb.201710095

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© 2018 Crown copyright. The government of Australia, Canada, or the UK ("the Crown") owns the copyright interests of authors who are government employees. The Crown Copyright is not transferable.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).

PMC Copyright notice

Figure 6. — Loss of ORP5/8 increases both PI4P and PI(4,5)P₂ levels in the PM. (A) PM PI4P levels in ORP5-, ORP8-, or ORP5/8-depleted cells. HEK293-AT1 cells were transfected with control, ORP5-, ORP8-, or both ORP5- and ORP8-silencing siRNAs for 2 d and subjected to BRET analysis monitoring PM PI4P. Three independent experiments performed in triplicate were each normalized to control. Grand means ± SEM are shown. Statistical analysis was performed by two-way ANOVA (**, P < 0.005; ns, not significant). (B) Same experiments as in A except that PM PI(4,5)P₂ levels were assessed by BRET measurements. (C) Effect of knockdown of ORP5/8 on PI4P levels in PIP5Kβ-expressing cells. HEK293-AT1 cells were transfected with the indicated siRNAs as in A. 1 d after siRNA transfection, cells were transfected with pcDNA3.1(HA) or myc-tagged PIP5Kβ (2 ng/well). 1 d after DNA transfection, cells were subjected to BRET measurement for PM PI4P. Statistical analysis from three independent experiments performed in triplicate was obtained as described for A. (D) Representative live-cell confocal images showing changes in PI4P distribution in response to PIP5Kβ expression in cells treated with control or ORP5/8-targeting siRNAs. HEK293-AT1 cells were transfected with indicated siRNAs. 1 d after siRNA transfection, cells were cotransfected with GFP-tagged P4M(2x) and mRFP-tagged PIP5Kβ (50 ng/well). 1 d after DNA transfection, cells were observed with a confocal microscope. Note the presence of some PI4P in the PM even in the PIP5K-expressing cells in the ORP5/8-depleted cells. Bars, 10 µm. (E) Kinetics of PM PI4P decrease after A1 treatment (left) in cells expressing PIP5Kβ with or without depletion of ORP5/8. Experiment was as described in C except for treatment of cells with A1 (30 nM) after a control measurement period. The rate of PM PI4P decrease was calculated for the time period indicated by the gray area (D). Grand means ± SEM are shown from three independent experiments performed in triplicate. Values were normalized to the rate value calculated for the vector-transfected cells treated with control siRNA. Statistical analysis was obtained with two-way ANOVA (**, P < 0.005).