Goat anti mouse igg hrp antibody

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).

HUMSCs, lysed in RIPA buffer, and frozen lung tissues, ground in a mortar with liquid nitrogen, were all stored aliquots at −80°C until assayed, respectively. Lysates were mixed with an equal volume of sample buffer, denatured by boiling, and then separated on a 10–15% polyacrylamide minigel. The proteins were transferred to nitrocellulose membranes, blocked with 5% milk, and incubated overnight with rabbit polyclonal anti-CXCR4 antibody (for HUMSC sample, 1 : 100, Boster, China, BA0761), mouse monoclonal anti-CXCR4 antibody (for lung tissue samples, 1 : 500, Abcam, USA, ab58176), or mouse monoclonal anti-TGF-β1 antibody (1 : 1000, Abcam, USA, ab64715) at 4°C. And then the blots were incubated with goat-anti-rabbit IgG HRP antibody (1 : 1000, Applygen Technologies Inc., China, C1309) or goat-anti-mouse IgG-HRP antibody (1 : 5000, Abcam, USA, A4416) for 1 h at room temperature. The membranes were rinsed with washing buffer and incubated with chemiluminescence (ECL) working solution for 5 min. The signals were detected by Gel software, and the relative intensities of the detected bands compared between groups.