Anti foxo3

To localize the expression of CHIT1, TGFBRAP1, and FOXO3, double-label IHC was undertaken with a modification of procedures described previously by our laboratory (Lee et al, 2016 (link)). NHLF cells (1 × 10⁶ cells/well) were cultured overnight in four-well chamber slides (Falcon) at 37°C in 5% CO₂ incubator, then washed with PBS. To unmask antigens, the slides were placed for 20 min at high temperature under high pressure in citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6). The slides were then blocked with a nonserum protein-blocking reagent (DakoCytomation Inc) for 1 h at room temperature and incubated with primary antibodies (anti-CHIT1 [1/50 dilution, AF-5325; R&D system], anti-TGFBRAP1 [1/50 dilution, SC-13134; Santa Cruz Biotechnology], and anti-FOXO3 [1/50 dilution, 12829S; Cell Signaling]) for 60 min at room temperature in a humid chamber. Substitution of the primary antibody with PBS served as a negative control. The slides were then washed in PBST (0.01% Tween 20) and incubated with secondary antibodies conjugated with horseradish peroxidase (Cell Signaling). Reaction products were developed using DAB containing 0.3% hydrogen peroxide up to 5 min. Cell nuclei were stained with mounting medium with DAPI (50 μl, H-1200; Vector Laboratories). Fluorescence was detected by Immunofluorescence microscopy.

The ChIP assay was performed as previously described^{33 (link)}, in BVES-KO skeletal muscles using the Magna ChIP A/G Chromatin Immunoprecipitation Kit (17-10085, Sigma-Aldrich). Soluble chromatin was co-immunoprecipitated with rabbit polyclonal anti-FoxO3 (1:200, #2497, Cell Signaling Technology) or an equal amount of control rabbit IgG (#2729, Cell Signaling Technology). After decrosslinking of the DNA, samples were subjected to quantitative PCR. The oligonucleotide primers used are shown in Supplementary Data 2.

After fixation, cochlear samples were blocked with 10% normal donkey serum in 10 mM phosphate-buffered saline (PBS, pH 7.4) with 0.3% Triton X-100 for 1 h at room temperature and then incubated with primary antibody overnight at 4 °C. The next day, the tissues were incubated for 2 h at 4 °C with 488- or 594-conjugated donkey secondary antibody (Invitrogen) and DAPI (Sigma-Aldrich). Omission of primary antibody served as the negative control. The following primary antibodies were used: anti-β-catenin (BD Biosciences), anti-myosin VIIA (Myosin7a) (Proteus Biosciences, Ramona, CA, USA), anti-cleaved caspase-3 (Cell Signaling Technology), anti-Foxo3 (Cell Signaling Technology), anti-Parvalbumin (Sigma-Aldrich), TUNEL (Roche, Indianapolis, IN, USA), and MitoSOX Red (Life Technologies, Rockford, IL, USA). Cochleae were dissected into apical, middle, and basal turns, and images were taken using a Leica SP5 confocal fluorescence microscope (Leica Microsystems, Biberach, Germany).