Anti foxo1

Protein levels in OVCA433 and OVCA429 cells were measured by western blotting. OVCA433 and OVCA429 cells were homogenized in RIPA buffer (150 mM sodium chloride, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 7.5, and 2 mM EDTA) containing proteinase inhibitor cocktail (Roche, Nutley, NJ) to lyse the cells. Then, cell lysates were centrifuged at 13,500×g for 30 min, and supernatants were recovered. Lysate supernatants containing about 30 μg of protein were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and analyzed by western blotting using anti-α-tubulin (mouse antibody clone# sc-5286; Santa Cruz Biotechnology, Santa Cruz, CA) and anti-FOXO1 (mouse antibody clone# sc-374,427; Santa Cruz Biotechnology) antibodies.

Human embryonic kidney 293 (HEK-293) cells and HepG2 human hepatoma cells were cultured in Dulbecco's modified Eagle medium and RPMI 1640 medium, respectively, supplemented with 10% fetal bovine serum (FBS) (Gibco Laboratories, Grand Island, NY, USA), 2 mM glutamine, penicillin (100 U/ml), and streptomycin (100 µg/ml) in a humidified 5% CO₂ atmosphere at 37 °C. Cryopreserved primary hepatocytes from HMGA1^–/– and wild-type mice [11 (link)] were cultured on matrigel-coated six-well plates in Williams E media (Sigma) supplemented with 10% FBS and then utilized in subsequent experiments. Cultured lymphoblast cell lines transformed with Epstein-Barr virus (EBV) were from normal volunteers and previously identified diabetic subjects carrying HMGA1 gene defects [30 (link), 34 (link), 40 (link)], and maintained in RPMI 1640 medium supplemented with 15% FBS. Nuclear protein extracts were prepared as previously [29 (link)] and final protein concentration in the extracts was determined by the modified Bradford method (Bio-Rad Laboratories, Hercules, CA, USA). The antibodies used for Western blot studies were: anti-HMGA1 [41 (link)], anti-FoxO1 (Santa Cruz Biotechnology, Santa Cruz, CA) and anti Sp1 [42 (link)].

Chromatin immunoprecipitation assays were performed with SimpleChIP enzymatic ChIP kit (Cell Signaling) with anti-FoxO1 (SantaCruz) as described [72 (link)]. The immunoprecipitated DNA subjected to RT-PCR analysis with iTaq Universal SYBR Green Supermix (Bio-Rad) using the primer (SABiosciences, catalog no. GPM1030787(-)02A) that amplify a region of the mouse irf4 promoter containing the predicted FoxO1 binding sites. Data were analyzed with the LightCycler 480 software. The results were normalized to control IgG and input DNA.

Liver and colon tissue were resected, homogenized, and centrifuged (15,000 rpm for 5 min). The protein supernatants were collected and then total protein was extracted with tissue protein extraction reagent (T‐PER, Pierce Biotechnology). Total protein content was determined using the BCA assay kit according to the manufacturer's protocol. The proteins in each sample were separated by SDS‐PAGE (10%–12% gel) and then transferred to a PVDF membrane (Millipore Corp.). PVDF membrane was sealed with 5% skimmed milk at room temperature for 1 h and incubated with primary antibody at 4°C overnight. After washing with Tris‐buffered saline (TBS) for three times, the membranes and corresponding secondary antibodies were incubated at room temperature for 1 h. The materials used included anti‐ZO‐1 (Sangon) and anti‐Claudin1, anti‐Sirt1, anti‐PGC‐1α, anti‐FoxO1, and corresponding secondary antibody (Santa Cruz Biotechnology).