Rabbit anti foxo1

Cells were plated at a density of 8000 cells/well in a 24-well plate and then washed with phosphate buffered saline (PBS), fixed with 4% paraformaldehyde treatment for 30 min, permeabilized in 0.5% Triton X-100 for 15 min, and incubated with 5% bovine serum albumin (BSA) for 30 min. Subsequently, cells were incubated with the antibodies in 5% BSA for 1 h at room temperature [rabbit anti-FOXO1 (Cell Signaling Technology)]. Afterward, cells were incubated with secondary antibodies for 1 h [goat anti-rabbit Alexa Fluor 488 (Cell Signaling Technology)]. After washing the cells with PBS, nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA), and samples were observed on a fluorescence microscope (Olympus).

Immunohistochemistry staining was performed by the Pathology Core at The Centre for Phenogenomics. Tissue sections were submitted to heat-induced epitope retrieval with citrate buffer (pH 6.0) or with TRIS-EDTA (pH 9.0) for 7 min, followed by quenching of endogenous peroxidase with Bloxall reagent (Vector). Non-specific antibody binding was blocked with 2.5% normal horse serum (Vector), followed by incubation for 1 h in Rabbit anti-PDX-1 (Abcam, ab47267, 1:400), or Rabbit anti-FOXO-1 (Cell Signaling Technologies #2880, 1:75). After washes, sections were incubated for 30 min with ImmPRESS HRP reagent Anti-Rabbit (Vector) followed by DAB reagent, and counterstained in Mayer’s hematoxylin. Densitometric analysis was performed using Image J.

Cellular protein was extracted for 10 min in 50 mM Tris-HCl buffer, pH 7.4, containing 0.5% NP-40, 0.7% NaCl, 0.2% EDTA, and protease inhibitors. Samples of 20–40 µg protein were resolved by SDS-PAGE and transferred to Immobilon-P Membrane (Millipore). Non-specific sites were blocked for one hour at room temperature in blocking buffer containing 5% skim milk in TTBS buffer. The membrane was then incubated with rabbit anti-β-catenin (1∶10000, Abcam), mouse anti-SNAI2 (1∶200, Abgent), rabbit anti-FOXO1 (1∶1000, Cell Signaling), or rabbit anti-phospho FOXO1 (1∶1000, Cell Signaling). The bound antibody was visualized with appropriate horseradish peroxidase-conjugated anti-IgG (Jackson) and SuperSignal West Chemiluminescent Substrate kit (Pierce). Signal intensity was quantitated using TINA software.

For immunofluorescence, HUVECs were seeded on gelatin-coated glass cover slips plated in 24-well plates and transfected with corresponding siRNA amount as described above. The cells were grown overnight in ECGM with 10% FCS. After incubation in 0.5% FCS for 24 h, the cells were washed with PBS and fixed with 4% formaldehyde for 10 min at room temperature. Then, cells were incubated in the blocking and permeabilization buffer containing 2.5% BSA and 0.3% Triton X-100. Subsequently, the cells were incubated with primary antibodies overnight at 4 °C. After washing with PBS, cells were incubated with secondary antibodies conjugated with FITC or TRITC for 1 h at room temperature. Finally, the slides were washed and mounted with mowiol (Calbiochem, Germany). The primary antibodies were rabbit-anti-FoxO1 (Cell Signaling), goat-anti-Ang-2 (Santa Cruz) and mouse-anti-O-GlcNAc (Abcam). The secondary antibodies were swine anti-rabbit FITC (DakoCytomation), swine anti-rabbit TRITC (DakoCytomation, Glostrup, Denmark), goat anti-mouse-FITC (Sigma-Aldrich) and Donkey anti-goat FITC (Acris, OriGene Europe, Herford, Germany). Photos were taken by confocal laser scanning microscopy (Leica Microsystems, Germany). Quantification of the protein expression in immunofluorescence was performed using Image J (NIH, USA).

Fifteen cochlear explants of each experimental group were lysed using an ultrasonic vibrator in 150 μl ice-cold Western Lysis Buffer (Beyotime, Nantong, China) for total protein extraction. Approximately 30 μg of total protein were fractionated by 8% SDS-PAGE and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). Target protein was probed with primary antibodies overnight at 4 °C. The primary antibodies were as follows: rabbit monoclonal anti-Bax (1:1000) (Cell Signaling Technology); rabbit monoclonal anti-caspase 9 (1:1000) (Cell Signaling Technology); rabbit monoclonal anti-caspase 8 (1:1000) (Cell Signaling Technology); rabbit anti-FOXO1 (1:1000) (Cell Signaling Technology); rabbit anti-FOXO3a (1:1000) (Cell Signaling Technology); and rabbit anti-GAPDH (1:5000) (Cell Signaling Technology). HRP-conjugated anti-rabbit IgG was used as the secondary antibody (1:5000) (Cell Signaling Technology). Bands were visualized using the ECL kit according to the manufacturer's instructions (Pierce, Thermo Scientific).

The following primary antibodies were used for Western blotting: rabbit-anti-Insulin receptor β (Santa Cruz, #711), rabbit-anti-phospho Akt (Ser473) (Cell Signaling, #4060), rabbit-anti-Akt (Cell Signaling, #9272), rabbit-anti Gsk3β (Cell Signaling, #9315), rabbit-anti-phospho Gsk3β Ser9 (Cell Signaling, #5558), rabbit-anti FoxO1 (Cell signaling, #2880), rabbit-anti-phospho FoxO1 Ser256 (Cell Signaling, #9461), rabbit-anti p70 S6 kinase (Cell Signaling, #2708), mouse-anti-phospho p70 S6 kinase Thr389 (Cell Signaling, #9206), mouse-anti S6 (Cell Signaling, #2317), rabbit-anti-phospho S6 Ser235/236 (Cell signaling, #4856), rabbit-anti 4E-BP1 (Cell Signaling, #9644), rabbit-anti-phospho 4E-BP Thr37/46 (Cell Signaling, #9459), mouse-anti-Gapdh (Abcam #ab8245), rabbit-anti-phospho HSL Ser660 (Cell Signaling, #4126), rabbit-anti-phospho HSL Ser563 (Cell Signaling, #4139), rabbit-anti-HSL (Cell Signaling, #4107), rabbit-anti-PLIN1 (Cell Signaling, #9349) and rabbit-anti-CD36 (Cell Signaling #14347). Secondary antibodies used for western blotting were goat-anti-rabbit IgG-HRP conjugate (Biorad, #1706515) and goat-anti-mouse IgG-HRP conjugate (Biorad, #1706516).