Nucleospin rna protein extraction kit

Western blots were carried out as previously described (Gibbs et al., 2011, 2014b). Anti‐HA antibodies were obtained from Sigma, and anti‐GUS antibodies were obtained from Invitrogen. Barley leaf samples (100 mg) were ground to powder with a mortar and pestle in liquid nitrogen, and protein extraction was performed (NucleoSpin^® RNA/Protein extraction kit, Macherey‐Nagel, Düren‐Germany). Total protein content in the samples was quantified by Bradford protocol with a spectrophotometer (Thermo Scientific Multiskan Ascent, Waltham, MA) against a BSA standard curve. For anti‐GUS Western blots, in each lane, 75 μg of total protein were loaded plus 5 μL of cracking buffer (0.5 m Tris–HCL, pH 6.8; 10% glycerol; 1% SDS; 5% β‐mercaptoethanol; 2 mg bromophenol blue) and water to a final volume of 20 μL. After 0.1% SDS–16.5% PAGE, proteins were electroblotted onto a nitrocellulose membrane. SeeBlue Plus2 Pre‐Stained Standard Marker (Novex, Paisley, UK) was loaded as a reference for protein size. Membranes were reversibly stained with Ponceau S Red to check equal loading and protein integrity.

Cells were preserved in RP1 lysis buffer complemented with β-mercaptoethanol (1%) until RNA and total proteins extraction using the NucleoSpin RNA/protein extraction kit (Macherey-Nagel). Supernatants from stimulated cells where concentrated using MeOH/chloroform. All RNA samples were treated with DNAse before reverse transcription was performed.

HEK293 cells (obtained from ATCC) were grown in DMEM supplemented with 10% foetal calf serum and GlutaMAX-1 (Invitrogen).
Stable cell lines were produced using the T-REx tetracycline-regulated expression system for mammalian cells (Invitrogen). Transfection of HEK293 cells was performed using FuGENE HD (Promega) using 2 µg of plasmid DNA and 5 µl of transfection reagent per well. For selection of stable cell lines, transfected cells were allowed to recover for 48 hours before selection with full growth medium supplemented with 5 µg/ml blasticidin (Invitrogen) and/or 200 µg/ml zeocin (Invitrogen). For infections, 10⁶ mouse macrophage RAW264.7 cells were seeded per well in a 12 well tray. Once the cells were attached, the cells were infected with MNV-CW1 at an MOI = 1. Cells were incubated with the virus at 37°C for 90 minutes and then the media changed. Time points were taken at 4, 8, 16, and 24 hours by scraping up the cells and then processing with NucleoSpin RNA/Protein extraction kit (Macherey-Nagel). Protein in a final volume of 100 µl was produced, the protein concentration quantified by BSA assay and equal quantities loaded onto 4–20% SDS-PAGE gels for analysis.