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

. 2017 Aug 1;28(16):2202–2219. doi: 10.1091/mbc.E17-01-0030

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

© 2017 Shiwarski et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

“ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.

PMC Copyright notice

FIGURE 5: — Class II PI3K C2A is required for surface trafficking of δR. (A) Immunoblotting for PI3K C2A (C2A) confirmed knockdown of C2A in PC12 cells stably expressing C2A lentiviral shRNA. Three different shRNA sequences were used to determine the most efficient knockdown. Representative immunoblot, with actin as a loading control. Blot densitometry was performed to quantitate the percentage of knockdown for C2A shRNA sequences 1–3 compared with control (Ctl) cells. (B) Densitometry (of three independent experiments) revealed C2A shRNA sequences 2 and 3 as providing significant reduction in C2A protein expression, with shRNA 3 providing the best knockdown at 49% (three independent experiments; mean ± SEM; **p < 0.01 by two-sided t test vs. Ctl). (C) Example images (of three independent experiments) for PC12 cells expressing FAP-δR (δR) with and without lentiviral PI3K C2A shRNA 3 (C2A shRNA) with a GFP reporter. Cells stably expressing the PI3K C2A shRNA were identified via GFP expression and had increased intracellular δR. (D) Image analysis and quantification revealed a significant increase in percentage of total δR fluorescence that overlaps with the Golgi in cells stably expressing the PI3K C2A shRNA compared with δR-only cells (δR, 102 cells; δR + C2A shRNA, 75 cells; mean ± SEM; ****p < 0.0001 by two-sided t test vs. δR). (E) Further quantification confirmed a significant increase in percentage of cells with Golgi-localized δR in cells stably expressing the PI3K C2A shRNA compared with δR-only cells (δR, 102 cells; δR + C2A shRNA, 75 cells; mean ± SEM; ****p < 0.0001 by two-sided t test vs. δR). (F) Example images from PC12 FAP- δR cells expressing EPAC cAMP biosensor and control nontargeted siRNA. Images reveal an increase in cAMP EPAC FRET ratio over time after 5 µM forskolin addition and subsequent decrease in cAMP and receptor internalization after δR agonist DADLE (10µM) addition. (G) Quantitative analysis of the EPAC FRET ratio, showing an increase in cAMP after forskolin addition and decrease after δR agonist DADLE addition in control (Ctl) siRNA cells. Similar experiments performed in cells transfected with PI3K C2A siRNA exhibited no decrease in cAMP after δR agonist DADLE (Ctl siRNA, 12 cells; C2A siRNA, 10 cells; mean ± SEM). (H) Analysis of percentage of cAMP inhibition after δR agonist addition demonstrates PI3K C2A siRNA ability to significantly reduce cAMP inhibition compared with control siRNA cells (Ctl siRNA, 12 cells; C2A siRNA, 10 cells; mean ± SEM; ****p < 0.0001 by two-sided t test Ctl vs. C2A siRNA).