Enhanced ecl luminescence detection kit

Total protein was extracted from the treated A549 and H1299 cells using RIPA Lysis Buffer (Vazyme, cat. no. FD008). And the concentration was then quantitated using Pierce BCA protein assay kit (Rockford). The equivalent proteins in each group were isolated using 10% SDS-PAGE according to their molecular weights, and the separate proteins were transferred to PVDF membrane (Millipore). After blocking with 5% skim milk for 2 h, the membranes were incubated with primary antibodies at 4 °C overnight, followed by second antibody (Abcam) for 1 h. Finally, the membranes were treated with Enhanced ECL luminescence detection kit (Vazyme; E411-04), and the results were examined on FluorChem™ M System. The primary antibody included anti-FOXO3 (Abcam, ab17026), anti-FOXO1 (Abcam, ab39670), anti-GRB2 (Abcam, ab111031), and anti-GAPDH (Abcam, ab37168).

The A. fumigatus conidia were cultured in liquid AMM in a rotary shaker at 200 rpm at 37°C for 48 h, followed by being exposed to 300 µg/mL CR for 0.5, 1, or 2 h. The mycelia were collected and ground in liquid nitrogen with a mortar and pestle. Total protein isolation was carried out in alkaline lysis buffer (0.2 M NaOH and 0.2% β-mercaptoethanol) as previously described (76 (link)). The lysate samples were loaded on 10% SDS-PAGE gel and blotted on a polyvinylidene difluoride membrane (Millipore) and then hybridized with anti-FLAG (Sigma-Aldrich, F3165) and anti-β-actin (ABclonal, AC026). The secondary antibodies were peroxidase-labeled goat anti-mouse (Proteintech, SA00001-1) and goat anti-rabbit (Proteintech, SA00001-2) and detected by an enhanced ECL luminescence detection kit (Vazyme) and then visualized by the imaging system (Bio-Rad). The band intensity was calculated using ImageJ software.