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Membranes were blocked using blocking solution for 30?min, washed, incubated for 1?h at room temperature with Mouse anti-STIM1 (1:500) (BD Biosciences), Rabbit anti-Orai1 (1:500) (Sigma-Aldrich), Mouse anti-GAPDH (1:1000) (AbD Serotec), pre-diluted Pierce Anti-Villin-2 mouse monoclonal antibody (Thermo Fisher Scientific, Hamburg, Germany) and bands were visualized by secondary antibody solution (Invitrogen). ImageJ software was used to perform densitometric analysis. Offline image analysis including brightness/contrast correction was performed with ImageJ software. If not mentioned otherwise, data are presented as mean?��?SD and represent at least three or more independent experiments. Ntotalcells represent total number of cells tested for a particular condition. Igor Pro 5.04 (WaveMetrics, Portland, OR, USA) was used for data analysis, curve fitting and graphs. http://www.selleckchem.com/screening/tyrosine-kinase-inhibitor-library.html Unpaired two-tailed Students t-test was used http://www.selleckchem.com/GSK-3.html to determine statistical significance (*) indicates P? http://en.wikipedia.org/wiki/VAV2 are shown as F/F0 (where F is the fluorescence intensity at the given time point and F0 is the fluorescence intensity at t = 0) for ER tracker-red, plotted over time in (B), (D), (F) and (H) for control, SLO, H2O2 and Triton X-100 respectively. Data are given as mean �� SD (n = 45 cells for control, n = 42 cells for SLO, n = 33 cells for H2O2 and n = 42 cells for Triton X-100 from three independent experiments. Fig. S3. No internalized SLO was detected in H441 cells. A. Concentration series of SLO.