IL-17A levels in conditioned medium from lung, CD3+ T cell cultures were determined by ELISA after 24 h stimulation (R&D Systems) according to the manufacturer’s instructions. Individual groups were tested in triplicate for technical replicates. Each experiment was repeated at least five times.
For Western blot, cell suspensions were harvested directly into pre-cooled Eppendorf tubes rinsed twice with ice-cold PBS. The collected cell suspensions were spun at 6,000 rpm for 2 min and supernatants were completely removed. Protein was extracted from each tube and entire contents resolved on 4–12% NuPage polyacrylamide gels essentially as described (Millar et al., 2020 (link)). Primary antibodies were incubated overnight at 4°C on blocked membranes. Antibodies not previously described include anti-phospho-STAT3 (1:2,000; #9145; Cell Signaling Technology), anti-STAT3 (1:1,000; #9139; Cell Signaling Technology), anti-phospho-p65 (1:1,000; #3033; Cell Signaling Technology), anti-p65 (1:1,000; #8242; Cell Signaling Technology), anti-phospho-FOXO1/FOXO3A (1:1,000; #9464; Cell Signaling Technology), and anti-FOXO1 (1:1,000; #18592-1-AP; Proteintech). HRP-linked secondary antibodies (Cell Signaling Technology) were used at 1:2,000 as described (Millar et al., 2020 (link)). Proteins were visualized by chemiluminescence using the ChemiDoc system (Bio-Rad).
For Western blot, cell suspensions were harvested directly into pre-cooled Eppendorf tubes rinsed twice with ice-cold PBS. The collected cell suspensions were spun at 6,000 rpm for 2 min and supernatants were completely removed. Protein was extracted from each tube and entire contents resolved on 4–12% NuPage polyacrylamide gels essentially as described (Millar et al., 2020 (link)). Primary antibodies were incubated overnight at 4°C on blocked membranes. Antibodies not previously described include anti-phospho-STAT3 (1:2,000; #9145; Cell Signaling Technology), anti-STAT3 (1:1,000; #9139; Cell Signaling Technology), anti-phospho-p65 (1:1,000; #3033; Cell Signaling Technology), anti-p65 (1:1,000; #8242; Cell Signaling Technology), anti-phospho-FOXO1/FOXO3A (1:1,000; #9464; Cell Signaling Technology), and anti-FOXO1 (1:1,000; #18592-1-AP; Proteintech). HRP-linked secondary antibodies (Cell Signaling Technology) were used at 1:2,000 as described (Millar et al., 2020 (link)). Proteins were visualized by chemiluminescence using the ChemiDoc system (Bio-Rad).
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