Akt and phospho akt antibodies

Akt and phospho-Akt antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). The GAPDH antibody was obtained from Kangchen (Shanghai, China). Goat anti-mouse IgG and anti-rabbit IgG were obtained from Calbiotech (San Diego, CA, USA). HUVECs were plated into a 6-well plate (3 × 10⁵ cells/well). After adhering, the cells were starved and incubated in serum-free medium for 24 h and then treated with VEGF₁₆₅ (20 ng/mL) in the presence or absence of AuNPs (125–250 nM) for another 24 h. Whole-cell lysates were collected and boiled for 10 min in 2x SDS sample buffer, subjected to 10% SDS-PAGE, and transferred to PVDF membranes (Amersham Life Sciences). The blots were blocked in blocking buffer (5% nonfat dry milk/1% Tween-20 in TBS) for 1 h at room temperature and then incubated with the primary antibodies (anti-Akt, 1 : 5000 dilution; anti-phospho-Akt, 1 : 500; and anti-GAPDH, 1 : 10,000) in blocking buffer for 2 h at room temperature. The bands were then visualized using horseradish peroxidase-conjugated secondary antibodies (1 : 2000) and ECL (Pierce Biotech, Rockford, IL, USA). The experiments were repeated three times.

Male Wistar rats (weighing ~250 g) and C57BL/6 mice (weighing 20 ± 2 g) were obtained from the Center of Comparative Medicine, Yangzhou University, China. The animals were housed at 22 ± 2 °C and under 55 ± 5 % relative humidity with a regular 12-h light/dark schedule. Food and water were available ad libitum.
Roxarsone (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 5 mL methanol and then diluted to 50 mL with deionized water to obtain a 1.0 mM stock solution. The working solution of 0.01–10.0 µM Roxarsone was made by further diluting the stock solution with incubation medium.
Sodium heparin and trypsin were purchased from Sigma-Aldrich. Dulbecco’s modified Eagle medium (DMEM), penicillin/streptomycin and fetal bovine serum (FBS) were purchased from Gibco (Invitrogen, Carlsbad, CA, USA). Recombinant rat VEGF₁₆₅ was purchased from Peprotech CO. (Rocky Hill, NJ, USA). The PI3K inhibitor LY294002 was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Growth Factor Reduced Matrigel™ Matrix was purchased from BD Biosciences (San Jose, CA, USA). Rabbit monoclonal PI3K, phospho-PI3K, Akt and phospho-Akt antibodies were from Cell Signaling Technology (Danvers, MA, USA). Rabbit polyclonal VEGF antibody, 5-bromo-2-deoxyUridine (BrdU) and mouse monoclonal BrdU antibody were purchased from Boster Biotechnology (Wuhan, China).

Protein expression in whole lungs was measured with immunoblotting as previously described [8] (link) using commercially available antibodies. The intensity of the bands was normalized to the intensity of a reporter protein (actin) using the Kodak Gel-doc system. Akt and phospho-Akt antibodies were obtained from Cell Signaling (Cat # 9272 and 9271, Danvers, MA), SIRT1 antibody from Santa Cruz Biotechnology Inc (sc-15404, Santa Cruz, CA), caspase 3 and CD31 from Abcam (ab13847 and ab24590, Cambridge, MA, USA).