Anti foxo1

Paraffin-embedded kidney tissue sections were dried, deparaffinized, and rehydrated. Following microwave pretreatment in citrate buffer (pH 6.0), the slides were immersed in hydrogen peroxide (3%) for 20 min to block the activity of endogenous peroxidases. Histopathological observations were performed with hematoxylin and eosin (H&E)-stained slides. Tissue sections were incubated overnight at 4°C with anti-LC3 (MBL, Japan), anti-FoxO1 (Proteintech Group, USA) or anti-NRF2 (Proteintech Group, USA) antibodies. The sections were then incubated with a secondary antibody for 1 h at room temperature, and the slides were developed using a STARR TREK Universal HRP detection kit (Biocare Medical, Concord, CA). Finally, the slides were stained using hematoxylin.

As soon as the cell culture complied with each experiment’s specifications, the lysis buffer for the RIPA assay was used to extract the total protein. The blot was then electrophoresed, incubated with 5% milk for two hours, and then incubated with primary antibody overnight at 4 °C.An enhancement chemiluminescence reagent (Thermo, USA) and a chemiluminescence imager (GE, USA) were then used to image the membrane after being incubated with the second antibody for 2 hours at room temperature.Antibodies used in present study were as follows: anti-PLOD3 (Abcam, ab89263, 1:1000); anti-cleaved Caspase 3 (CST, 9661 T, 1:1000); anti-PCNA (CST, 13110 T, 1:1000); anti-CCND1 (CST, 55506 T, 1:1000); anti-MMP9 (CST, 2270 S, 1:1000); anti-MMP3 (CST, 14351 S, 1:1000); anti-BCL2 (CST, 4223 T, 1:1000); anti-FOXO1 (CST, 2880 T, 1:1000); anti-FOXO3 (2497 S, 1:1000); anti-FOXO4 (CST, 2499 S, 1:1000); anti-FOXO6 (Proteintech, 19122- 1-AP, 1:500); anti-p21 (CST, 2847 T, 1:1000);anti-ubiquitination (CST, 58395 S, 1:1000); anti-Survivin (CST, 2808 T, 1:1000); anti-β-Actin (CST, 4970 T, 1:1000).

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).