 | Figure 2
Nature Cell Biology
6, 1129 - 1134 (2004)
Published online: 24 October 2004; | doi:10.1038/ncb1188
Nitric oxide induces coupling of mitochondrial signalling with the endoplasmic reticulum stress responseWeiming Xu, Lizhi Liu, Ian G. Charles
& Salvador Moncada | | | | Figure 2. Calcium dependence of NO-mediated ATF6 cleavage and upregulation of Grp78.
(a) Western blot analysis showing ATF6 cleavage and Grp78 upregulation in NO-generating EcR293 clone-11 subclone-1 cells suspended in normal or calcium-free medium and treated with A23187. Cells shown in lane 1 (-NO) were not treated with muristerone A (n = 3). (b) Immunoblot analysis of calcium-dependent cleavage of Flag-tagged ATF6 in cell-free extracts in vitro. Top, insoluble membrane pellet fraction; bottom, soluble fraction after immunoprecipitation (IP) with Flag antibody. (c) The role of S1P and S2P in NO-mediated cytoprotection. EcR293 clone-11 subclone-1 cells were transfected with siRNA duplex (200 nM per well, 12 wells) on day one and day two. Cells were then treated with muristerone A and thapsigargin and the viability determined. All values are the mean values + s.d. of three replicates. Double asterisk indicates a significant difference (P < 0.01) between no siRNA treatment and either S1P siRNA-treated or S2P siRNA-treated samples. Bottom, real-time RT−PCR quantification using the total RNA prepared from cells treated with target-specific siRNA, sense siRNA, and non-silencing (NS) siRNA for 48h. GAPDH was used as an internal control. The values represent the amount of mRNA relative to that in the untreated cells (arbitrary value = 1). Asterisk indicates a significant difference (P < 0.05) between siRNA-treated and non-silencing siRNA-treated samples.
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