Foxo3a antibody

Tumor tissues from flank tumors and whole brain tissues from P7, adult Ptch1^+/+Trp53^-/-, and adult Ptch1^+/-Trp53^-/- mice were fixed in 4% formaldehyde overnight and standard paraffinization was performed. Sections were cut to 5 um thickness. Sections were rehydrated in xylene and serial ethanol concentrations. Antigen retrieval was achieved with sodium citrate at sub-boiling temperature in the microwave for 30 minutes. Sections were incubated overnight at 4°C with Foxo3a antibody (Cell Signaling, D19A7) or hexokinase 2 antibody and subsequently with anti-rabbit biotinylated secondary antibody (Vector Labs, BA-1000) for 30 minutes at room temperature. For hexokinase 2, sections were incubated with avidin/biotin ABC complex (Vector Labs, PK-6102) and stained with DAB substrate chromogen (DAKO, 2016–10). For Foxo3a, after secondary antibody incubation, sections were incubated with fluorescein-labeled avidin D for 30 minutes (Vector Labs). Slides were then mounted with Vectashield DAPI mounting medium for immunofluorescence (Vector Labs). High magnification images of sections were captured using a Nikon 90i microscope equipped with Roper EZ monochrome and DS-Fi1 color cameras. NIS Elements BR 3.0 software was used to capture and analyze the images.

Cell samples were collected and lysed in RIPA buffer (150 mmol/L sodium chloride, 0.5% sodium deoxycholate, 9.1 mmol/L dibasic sodium phosphate, 1.7 mmol/L monobasic sodium phosphate, 1% Nonidet P-40, 0.1% sodium dodecylsulfate(pH adjusted to 7.6) containing fresh protease inhibitor cocktails (Sigma-Aldrich, St. Louis, MO, USA).
Lysates were cleared by centrifugation. Then, equal amounts of protein were separated on an 10% SDS-Tris/glycine gel using mini-PROTEIN 3 Gel electrophoresis system (Bio-Rad, USA). After that, proteins were transferred to Polyvinylidene Fluoride Membrane (Millipore,USA),and the membrane was blocked with 5% milk power in TBS plus 1% Tween 20. The membranes were incubated with FoxO3aantibody (12829, Cell signaling technology, USA), c-Myc antibody (sc-40, Santa Cruz Biotech, USA) or actin antibody (PAB0174,Abnova,USA) overnight at 4°C. Immune complexes were detected with HRP-conjugated secondary antibodies and visualized by chemiluminescence reagents (ECL, Amersham Biosciences).

The human thyroid cancer cells were seeded onto glass coverslips in 6-well plates and cultured until 50-60 % confluence, followed by treatment with 200 nM NVP-BEZ235 for 48 h. Cells were then fixed with 4.0 % paraformaldehyde for 20 min at room temperature and blocked in 5.0 % goat serum for 30 min. Next, the coverslips were incubated at 4 °C with primary FOXO3a antibody (#2497, Cell Signaling Technology,) overnight. Subsequently, the cells were washed in PBS. The coverslips were incubated with goat anti-rabbit IgG H&L (FITC, ab6717, Abcam, San Francisco, CA, USA) for 1.5 h and dyed with Hoechst33342 (Beyotime biotechnology, Shanghai, China). The images were obtained with an Olympus IX71 microscope (Olympus) and color mergence was performed using ImageJ software (ImageJ version 1.44p, NIH, MD, USA).