Prior to immunofluorescence staining cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100/phosphate-buffered saline and blocked with 10% goat serum/phosphate-buffered saline. To visualize the ER chicken anti Bip IgY28 (link) was used as primary and goat anti-chicken IgG conjugated to Alexa647 (Life Technologies) as secondary antibodies. FLAG-tagged CALR variants were detected using mouse monoclonal anti-FLAG as primary (Sigma-Aldrich, St Louis, MO, USA), and goat anti mouse IgG conjugated to Alexa Fluor488 (Jackson Immuno Research Laboratories, West Grove, PA, USA) as secondary antibodies. Nuclei were counterstained with Hoechst 33342 (2μg/ml in phosphate-buffered saline). The stained samples were analysed by the laser scanning confocal microscopy system (LSM 780; Carl Zeiss, Jena, Germany) with a Plan-Apo-chromat × 60 oil immersion lens (NA 1.4).
Materials and methods on the KISMET method (cell lines, kinase inhibitors, statistics, single-dose–response values, combination of two off-target data sets, selection of kinases used for KISMET and algorithm for the calculation of the kinase rank) can be found in theSupplementary Information .
Materials and methods on the KISMET method (cell lines, kinase inhibitors, statistics, single-dose–response values, combination of two off-target data sets, selection of kinases used for KISMET and algorithm for the calculation of the kinase rank) can be found in the
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