Complete protease inhibitors cocktail tablets

The pQCXIP vectors (Clontech) expressing the full-length wildtype and mutant SRP54 alleles (Carapito et al., 2017b) as well as the extracellular part of the non-conventional Major Histocompatibility Complex class I MICA protein (MICA*008 allelic variant) were previously reported (Carapito et al., 2017a) . Both vectors were transiently co-transfected in equal quantities (1mg) into HEK293 cells (Sigma-Aldrich) grown in suspension in serum-free Freestyle medium (Thermo Fisher Scientific) at 37 C, 8% CO2, 70% humidity, 100 rpm (50 mm shaking amplitude on an ISF1-X Shaker, Kuehner). For each transfection 2 x 10 6 cells were transfected with 7mg Polyethylenimine (PEI, Clinisciences) as transfection agent, i.e. a DNA/transfectant ratio: 1/3.5). The empty pQCXIP vector and a SRP54 variant with a neutral amino-acid modification (p.Ala4Gly) were used as controls. Cells and the cell culture supernatants were harvested 48 hours post-transfection. Cells were lysed using lysis buffer (20 mM Tris, pH7.5, 150 mM NaCl, 1% NP40 and protease inhibitors (complete protease inhibitors cocktail tablets, Roche)). The proteins were detected by Western blotting using a monoclonal anti-SRP54 antibody (SC-393855, Santa Cruz), an anti-GAPDH antibody (MAB374, Millipore), and an in-house developed mouse monoclonal anti-MICA antibody (Carapito et al., 2017a) .