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Figure 1. Generation and characterization of stable DT40 cell lines expressing Ins(1,4,5)P3 receptor phosphorylation mutants. (A) Cartoon representation of Ins(1,4,5)P3 receptor structure with sequence alignment around T-930, for the rat (rIP3R), human (hIP3R) and Xenopus (xIP3R) Ins(1,4,5)P3 receptors. (B) Ins(1,4,5)P3R expression levels in the different cell lines. Different cell lines were generated using the DT40 cell line where all three Ins(1,4,5)P3R isoform are deleted (KO). The 3KO cells were electroporated with the linearized plasmid for either the T930A or T930E mutants. The plasmid carrying the mutants contains G418 resistance. Stable cell lines for each mutant were established using G418 selection. WT: DT40 cell line expressing only the wild-type Ins(1,4,5)P3 receptor (type 1); T930A and T930E: expressing the respective mutant Ins(1,4,5)P3Rs. (C) Growth rates of the different cell lines are also equivalent. The same number of cells was plated at time zero and cells were counted at different time points under equivalent culturing conditions to determine whether the expression of the different mutants affects cell growth. Although the T930E mutant grew at a slightly slower rate the difference were not significant. (D) Resting Ca2+ levels and store Ca2+ content were measured in DT40 cells loaded with Fura-2 and incubated in Ca2+-free solution. Ionomycin was added as indicated to release Ca2+ from stores. Image published in: Haun S et al. (2012) Copyright © 2012 Landes Bioscience. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license Larger Image Printer Friendly View

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