SEARCH

SEARCH BY CITATION

FilenameFormatSizeDescription
jcmm1592-sup-0001-FigS1.tifimage/tif935KFig. S1 (A) WESTERN BLOT showing effectiveness of Orai1 shRNA knockdown strategy. Bands were resolved in 5–20% tris-glycine gels and evidenced using Orai-1 primary antibody (1:500, Santa Cruz) followed by secondary anti-rabbit antibody (1:1000, Santa Cruz). (B) Bar graph summarizing effects of overexpression of Orai3 or (C) Orai3 + STIM1 in NCM460 cells. Cells expressing an Epac-based cAMP sensor plated onto glass coverslips were co-transfected with Orai3 or Orai3 + STIM1 together with mCherry (used as marker of transfected cells; 300 ng each). Forty-eight hours after transfection the coverslips were mounted in a home-built flow through perfusion chamber and imaged using a 60× oil immersion objective. Overexpression of Orai3 or Orai3 + STIM1 did not significantly alter ionomycin-induced cAMP production compared to control cells.
jcmm1592-sup-0002-FigS2.tifimage/tif1318KFig. S2 (A) STIM2 is not required for store-operated cAMP signaling. Validation of three different strategies used for STIM2 silencing: (i) Dharmacon SmartPool (siRNA STIM2-a), (ii) Ambion silencer (siRNA STIM2-b), and (iii) Origene shRNA (shRNA STIM2). NCM460 or HeLa cells were transfected with 100nM siRNA or with 0.375 ng/μl of a plasmid expressing a single shRNA. Forty-eight hours after transfection, cells were lysed in RIPA buffer (Sigma-Aldrich) complemented with protease inhibitors (Sigma-Aldrich). Five to 10 μg of total cell lysate were resolved in 5–20% tris-glycine gels and electroblotted in PVDF membranes (Amersham Biosciences). The membranes were incubated for 2 hrs at room temperature with an anti-STIM2 polyclonal antibody (ProSci) diluted 1:500 in 5% milk/TBST. After washing with TBST membranes were incubated for 1 hr with secondary antibodies (1:2000; Santa Cruz). Peroxidase activity was detected with the enhanced chemiluminescence kit (Amersham Biosciences, ECL advance western blotting). Loading control, Glyceraldehyde-3-phopshate dehydrogenase (GAPDH; Santa Cruz 1:2000). (B) NCM460 cells expressing an Epac-based cAMP sensor plated onto glass coverslips were transfected with 100 nM of siRNAs against STIM2 (siRNA-a or siRNA-b) or their respective scramble siRNAs. Alternatively cells were co-transfected with shRNAs targeting STIM2 (or non-effective scramble) together with mCherry (used as marker of transfected cells). Forty-eight hours after transfection the coverslips were mounted in a home-built flow through perfusion chamber and cells were imaged using a 60× oil immersion objective. Initial experiments using a pool of four distinct siRNAs against STIM2 (siRNA-a, B-first bars) suggested that STIM2 attenuates SOcAMPs (data from 33 cells of scramble in 6 experiments; 73 cells siRNA STIM2 in 7 experiments; P < 0.0005). However, the scramble control was not typical, thus this series of experiments was inconclusive. Numerous subsequent experiments using either the same siRNA pool (siRNA-a) (data from 21 cells of scramble in 4 experiments; 16 cells siRNA STIM2 in 3 experiments) or two alternative strategies, a single siRNA (siRNA-b) (data from 18 cells of scramble in 4 experiments; 18 cells siRNA STIM2 in 4 experiments) or a single shRNA against STIM2 (shRNA-C) (data from 10 cells of scramble in 4 experiments; 5 cells siRNA STIM2 in 4 experiments) led to the conclusion that STIM2 is not a significant constituent of the SOcAMPs machinery in NCM cells.
jcmm1592-sup-0003-FigS3.tifimage/tif655KFig. S3 Overexpression of AC3 (n = 3 expts, 3 control cells and 9/11 mCherry cells), AC5 (n = 3 expts, 12 mCherry cells and 21 control cells), or AC6 (n = 3 expts, 27 control cells and 8 mCherry cells) in HeLa cells did not generally rescue the SOcAMPS phenotype. Shown is a typical result for AC3 overexpression. HeLa cells stable expressing Epac-H90 cyclic AMP based sensor were transfected the day after plating with the 300 ng AC isoform of interest plus 300 ng of mCherry plasmid. Experiments were performed 48 hrs after transfection.
jcmm1592-sup-0004-FigS4.tifimage/tif713KFig. S4 Left panel: Summary of data from Fig 4C and D in main text. Addition of PMA alone to NCM460 cells caused a minor increase in the FRET ratio (expressed as a percentage of the maximal response following treatment with forskolin + IBMX; from experiments like Fig. 4C in main text). Addition of PMA after ionomycin caused a minor elevation in the FRET ratio compared to ionomyicn alone (refer to Fig. 4D in main text). Right panel: Amplitude of ionomycin response (expressed as a percentage of the maximal FRET ratio change following treatment with forskolin + IBMX) after 15 min. and 3 hrs treatments with PMA.

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.