Ab181602

BMDMs were plated at 2 × 10⁶ cells/well in non-treated six-well cell culture plates, challenged with A. fumigatus at MOI of 5 and plates were subsequently spun at 400g for 2 min. After incubation for 2 h, cells were washed with PBS and put on ice. Cells were harvested and centrifuged at 1000 g for 10 min at 4°C and frozen in liquid nitrogen. After cells were quickly thawed at 37°C, samples were dissolved in PBS containing cOmplete Ultra Protease Inhibitor Cocktail (Roche) and homogenized with a 50-μl Hamilton syringe (Sigma, 58382) by taking up and expelling the suspension several times until it appeared as a homogeneous solution. Homogenate was kept on ice for the immediate measurement enzymatic activities. Enzymatic activity of respiratory chain complexes was performed as previously described with a spectrophotometer Spark plate reader (26 (link)). Cell homogenates were also used to evaluate the abundance of proteins in BMDMs treated with swollen conidia of A. fumigatus. Extracted proteins were analyzed by western blot with anti-GAPDH (Abcam, ab181602), anti-GLRX1 (R&DSystems, AF3119), anti-SOD2 (Abcam, ab13533) or NOX2 (Abcam, ab129068) antibodies.

For Western-blots, cells were lysed in RIPA buffer (#SLCD5849, Sigma, USA) and the protein concentration of each sample was determined by BCA protein assay kit (#XI357440, ThermoFisher Scientific, Waltham, USA). The protein concentration was adjusted to 20 µg of total protein which were denatured (10 min, 95^oC), and applied to electrophoresis was for size fractionation (110 V, open Amp, 50 min, at 4^oC) in a 4–12% SDS–PAGE (#M41212, GeneScript, Piscataway, USA). Proteins were then transferred onto a nitro-cellulose membrane (#88,018, ThermoFisher, USA) by heat-accelerated capillary transfer and over-night incubation at 50^oC. Primary antibodies were applied overnight at 4^oC, followed by visualization with secondary, horse radish labeled antibodies. The following antibodies were used: c-Jun (1:1000, Abcam ab40766); NF-kB p65 (1:400, Cell Signaling D14E12); GR (1:1000, Abcam ab183127); HDAC2 (1:2000, Abcam ab219053); HDAC3 (1:500, Abcam ab32369); HDAC5 (1:1000, Abcam ab55403); HDAC8 (1:1000, Invitrogen PA5-83916); GAPDH (1:1000, Abcam ab181602); α-tubulin (1:1000,R&D systems MAB9344); Anti-Rabbit IgG (1:2000, Sigma A9169-2ML); Anti-Mouse IgG (1:2000, Sigma A9917-1ML).
Blots were then quantified by the Image J (1.53 version).