Incubations with primary anti bodies were followed Pacritinib order by secondary labelling using sheep anti mouse HRP or goat anti rabbit. SuperSignal West Pico Chemi luminescent Substrate Inhibitors,Modulators,Libraries was used according to the manufacturers instructions for detection. Membranes were stripped between antibody staining procedures in Restore Western Blot Stripping Buffer for 15mins at 37 C. Murine anti tubulin or anti tubulin, murine anti GAPDH or Inhibitors,Modulators,Libraries rabbit anti B actin were used for loading controls. Active Ras Pull Down and Detection Cells were grown so they were sub confluent in T75 flasks prior to harvesting, processing and western blot ting for Ras small GTPase activation using the Active Ras Pull Down and Detection Kit. Experiments were performed per protocol according to the manufacturers instructions.
One step viral growth assays Inhibitors,Modulators,Libraries Cells were seeded at 1��105 in 24 well plates and treated on at 37 C with plating media alone, or plating media containing EGFR ligandinhibitors. The following day cells were infected with reovirus for 2 hrs. Monolayers were washed once with PBS and the ligandinhibitors replaced. Cells were scraped into the supernatant and harvested at time points post infection, freeze thawed three times and titred by TCID50 assay on L929 cells, as described previously. p38MAPK ELISA Cells were plated at 5 105 in 6 cm dishes. Cells were treated with SB202190 for 2 hours, harvested, and analysed for phospho p38 immunoassay SUV869, R D Systems, Minneapolis, USA. Experi ments were performed according to the protocol pro vided for the assay by the manufacturers.
Interferon ELISA Cal27, HN3, HN5 and SIHN 5B cells plated at 1 106 in 10 cm dishes were treated with reovirus at an MOI of Inhibitors,Modulators,Libraries 5, or left untreated. Cells were incubated for 24 hours and supernatants were collected and spun down to re move cell debris. Samples were stored at ?20 C until analysis for alpha, beta and gamma interferon by ELISA. IFN was analysed using match paired antibodies from Mabtech, IFN with match paired antibodies from BD Biosciences and IFN B using a kit from PBL Interferon Source according to the manufacturers instructions. Data were read on a Multiskan EX plate reader at 405 nm using Ascent software. JAM 1 FACS Analysis Cells were harvested with trypsin, pelleted and resus pended in FACS buffer. 1 105 cells in 100 uL were stained with 2 uL of JAM A antibody or isotype control and incubated for 30 minutes at 4 C.
One millilitre of FACS buffer was added and cells were pelleted. Pellets were either Inhibitors,Modulators,Libraries resuspended in 500 uL PBS and analysed within an find FAQ hour using a FACSCalibur machine, or fixed in 1% paraformaldehyde for analysis within 5 days. Statistics The data on EGFR status and reovirus cell killing were not normally distributed. Therefore Spearmans rank correlation was used to test the correlation between EGFR status and reovirus cytotoxicity.