Western blot analysis

Prestimulated human NK cells were coincubated with or without hyphae of A. fumigatus for 4 hours. NK cells at time point 0 hours served as control. Cells were lysed with RIPA Lysis and Extraction Buffer Kit (Thermo Scientific) containing HALT Phosphatase and Protease Inhibitor Cocktails (both Thermo Scientific). Proteins were separated by SDS-PAGE using 4-15% Mini Protean TGX Precast Protein Gels (Bio-Rad). Proteins were blotted onto a nitrocellulose membrane which was blocked with 5% skim milk-PBS solution. Mouse monoclonal antibodies against human IFN-γ (1:200; LifeSpan BioSciences, Seattle, USA), human perforin (1:1000, LifeSpan BioScience), human GM-CSF (1:1000, R&D Systems), and human GAPDH (1:20000; Biolegend) were used as primary antibodies, goat anti-mouse IgG-HRP antibody (1: 100000; Abcam, Cambridge, UK) as secondary antibody. For visualization, Gel Doc™ XR+ System (Bio-Rad) and Amersham ECL Select Western Blotting Detection Reagent (GE Healthcare Life Sciences, Little Chalfont, UK). For comparison of intracellular protein levels, the ratio of the protein of interest and GAPDH was calculated by determination of relative band intensities using Image Lab 5.0 software (Bio-Rad).