Image lab 4.1 analysis software

MDMs (5 × 10⁵/well, in 24-well plates) were infected with A. fumigatus conidia for 2 h at a 1:10 effector-to-target ratio at 37°C in 5% CO₂. After infection, cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris, 250 mM NaCl, 2 mM EDTA, 1% NP-40, 10% glycerol [pH 7.2], and a mixture of protease inhibitors [Roche Molecular Biochemicals]). Cell lysis was performed at 4°C for 30 min (with shaking), and samples were then centrifuged. The protein content was determined using the Bradford dye-binding (Bio-Rad) method. Laemmli buffer (Bio-Rad) was added to 20 μg of protein, and samples were boiled and separated on a 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAG) and transferred to nitrocellulose membranes (Bio-Rad). Western blotting was performed according to the manufacturer’s instructions, using the following primary antibodies: rabbit anti-PFKFB3 and mouse anti-β-actin, both from Abcam and diluted 1:1,000. Secondary antibodies used were anti-rabbit (Thermo Fisher Scientific) and anti-mouse (Bio-Rad), both diluted to 1:5,000. The blots were developed using chemiluminescence (SuperSignal West Femto maximum sensitivity substrate; Thermo Fisher Scientific) and detected with ChemiDoc XRS system (Bio-Rad). Signal intensities and quantifications were determined with the ImageLab 4.1 analysis software (Bio-Rad).

Western blot 24 h after stress treatment human KCs were washed twice with PBS and then harvested with lysis buffer (70 mM Tris-HCl, pH6.8, 1,1%SDS, 11,1% (v/v) glycerol, 0005% bromophenol blue (BioRad)) containing protease inhibitor cocktail (Abcam, Cambrige, UK) and Pierce TM Phophatase Inhibitor Mini Tablets (Thermo Fisher Scientific, Waltham, MA) on ice and immediately sonicated. Immunoblotting using antibodies for CDK1 (1:1000; ab32384, Abcam), AKR1C1 (1:1000; ab192785, Abcam), HMOX1 (1:1000, ADI-SPA-896, Enzo), and GAPDH (1:2000; clone 5G4; HyTest Ltd., Turku, Finland), was performed as previously described [34] (link). As secondary antibody, goat anti-rabbit IgG-HRP (Biorad 170–6515) or sheep anti-mouse IgG-HRP (NA-931-V, GE Healthcare, Little Chalfont, UK) were used and subsequent chemiluminescent quantification on ChemiDoc imager (Bio-Rad) was performed. The signal was measured with Image Lab 4.1 analysis software (Bio-Rad) and target bands were normalized to GAPDH.