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 Nov 15;8:1505. doi: 10.1038/s41467-017-01644-8

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

© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

PMC Copyright notice

Fig. 1 — Endogenous IP₃R1s form puncta. a In-gel fluorescence of lysates from EGFP-IP₃R1 HeLa cells (GR) and control (WT) cells demonstrates that the only fluorescence is associated with EGFP-IP₃R1 (green arrow). Results typical of four gels. Positions of selected M _r markers (kDa) are shown (a, c, d). b TIRFM images of EGFP-IP₃R1 HeLa cells showing a marker for the ER lumen (mCherry-ER). The merged image and an enlargement of the boxed area show co-localization of EGFP-IP₃R1 with mCherry-ER (Pearson’s coefficient with Costes’ automatic threshold = 0.93 ± 0.02; Costes P value = 1.00, n = 4 cells). Scale bar = 5 µm (2 µm for enlargement). c Western blots (WBs) for IP₃R1-3 show expression of tagged (green arrow, ~290 kDa) and untagged (black arrow, ~260 kDa) IP₃R1 in GR and WT cells, respectively. Expression of IP₃R subtypes in GR cells is shown relative to control (WT) cells (%, mean ± SD, n = 3 for IP₃R2 and IP₃R3, n = 4 for IP₃R1). Comparisons of band intensities using paired Student’s t-tests indicated no significant differences between WT and EGFP-IP₃R1 cells. d WB (IP₃R1-3 antibodies) from lysates of EGFP-IP₃R1 HeLa cells after immunoprecipitation with GFP-Trap. Eluate lanes were loaded with sample equivalent to 1.5 times the amounts loaded in the lysate lanes. Numbers show % of each subtype detected in the pull-down (n = 2). e Photobleaching of a punctum showing the final bleaching step (bracket) and the initial fluorescence (dashed line) used to calculate the total number of fluorophores (n). FU, fluorescence units. f Single-step photobleaching results (284 puncta from five cells, Supplementary Fig. 5) were used to calculate the number of tetrameric IP₃Rs per punctum (8.4 ± 7)