Anti pten antibody

Western blot analysis was conducted using standard methods. Proteins were separated on a 10% SDS-PAGE gel and then transferred to PVDF membranes (Amersham, Buckinghamshire, UK). The membranes were blocked overnight using 5% non-fat dried milk and incubated for 2 h with an anti-PTEN antibody (Abcam, Cambridge, UK; 1:1000), anti-AKT antibody (Bioworlde, Minneapolis, MN, USA; 1:1000), anti-p-AKT antibody (Bioworlde, Minneapolis, MN, USA; 1:1000), or anti-GAPDH antibody (Proteintech, Chicago, IL, USA; 1:50,000). After washing with TBST (10 mM Tris, pH 8.0, 150 mM NaCl, and 0.1% Tween20), the membranes were incubated for 2 h with a goat anti-rabbit antibody (ZSGB-BIO, Beijing, China; 1:5000 or 1:50,000).

The prostate cancer tissues samples (1.5 cm × 1.5 cm x 0.3 cm) were fixed in 10% formalin for 10 min, dehydrated in a gradient of ethanol for 2 h, and then transparent, paraffin and embedded. The slides (4-7 μm) were deparaffinized, rehydration and antigen-retrieved, after which slides were blocked by 3% H₂O₂ for 10 min and incubated with anti-FAM46C (Abcam, Cambridge, MA, USA) or anti-PTEN antibody (Abcam) at 25°C for 1 h and then stained with horseradish peroxidase (HRP)-labeled IgG (Shanghai Long Island Biotec. Co., Ltd, China) at 25°C for 20-30 min. Subsequently, the sections were stained with diaminobenzidine (DAB), counterstained with hematoxylin for 3 min and washed in water for 10 min. The tumor cells with positive staining more than 25% were defined as FAM46C or PTEN high expression group and that less than 25% were defined as FAM46C or PTEN low expression group.

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), bovine calf serum (BCS), penicillin and streptomycin mixture were obtained from Gibco (Grand Island, NY, USA). 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), dimethylsulfoxide (DMSO), 3-isobutyl-1-methylxanthine (IBMX), dexamethasone (DEX), and insulin were purchased from Sigma Aldrich (St Louis, MO, USA). Free fatty acid, adiponectin, and leptin Enzyme-linked immunosorbent assay (ELISA) kits were purchased from YiFeiXue Biotechnology (Nanjing, China) and Jian-Cheng Biotechnology (Nanjing, China). GLUT4 rabbit antibody, anti-PTEN antibody, anti-Fatty Acid Synthase antibody and Wortmannin were purchased from Abcam Biotechnology (Cambridge, UK). Total Akt rabbit polyclonal antibody and p-Akt-ser473 rabbit polyclonal antibody were supplied by Santa Cruze (California, USA). Goat anti-rabbit IgG (H + L) dylight 488 was supplied by Bioworld (Nanjing, China). Total PI3K rabbit polyclonal antibody, p-PI3K rabbit polyclonal antibody, total IRS1 rabbit polyclonal antibody, p-IRS1-ser307 rabbit polyclonal antibody, Na–K-ATPase rabbit antibody, β-actin rabbit polyclonal Antibody, and goat anti-rabbit IgG antibody were all provided by Nanjing Enjing Biotechnology (Nanjing, China).

Lung tissues were fixed using 4% paraformaldehyde and were serially dehydrated, permeabilized and embedded in paraffin. The tissues were cut into 5-μm sections and mounted on slides. Slides were incubated at 60°C for 3 h, and twice dewaxed in xylene and serially rehydrated in ethanol. Slides were washed with PBS and stained with Mayer's hematoxylin and eosin (HE). To immunohistochemically detect α-SMA, CD38 and PTEN, slides were subjected to antigen retrieval in citrate buffer for 15 min, followed by cooling for 40 min at room temperature. Tissue sections were incubated using 3% hydrogen peroxide for 15 min at room temperature. Then, tissue sections were incubated with goat serum for 30 min at room temperature, and incubated with primary antibodies including the anti-α-SMA antibody (1:100; Abcam), anti-CD38 antibody (1:200; Abcam) and anti-PTEN-antibody (1:50; Abcam) overnight at 4°C. Sections were then incubated with the HRP-conjugated secondary antibody for 20 min at room temperature. Sections were washed with PBS and stained with diaminobenzidine (DAB) for color development. The sections were observed, and images were acquired using an Olympus BX5 biological microscope (Olympus Corporation).

The concentration of proteins purified from LV or cultured NRVCs was determined with BCA Protein Assay Kit. The samples were subjected to electrophoresis in 10% SDS‐PAGE and then transferred to nitrocellulose filter membrane. The membrane was blocked in 5% skim milk at 25°C for 1.5 hours and then incubated with the primary antibodies. The anti‐PTEN antibody (1:1000; Abcam), anti‐phospho‐Akt (Ser473) antibody (1:2000; Cell Signaling), anti‐Akt (pan) antibody (1:1000; Cell Signaling) and GAPDH (1:2000; Proteintech) were used in this study. After washing four times with PBS, the membrane was incubated with the fluorescence‐conjugated secondary antibody (Invitrogen) at 1:8000 dilution for 1 hour. Western blot bands were quantified by using Odyssey infrared imaging system (LI‐COR).

L-Nitro-arginine-methyl-ester (L-NAME) was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany; batch no. N5751-10), and sodium pentobarbital was purchased from Shanghai Chemical Reagent Co., Ltd. (Shanghai, China). Anti-HIF-1 alpha antibody, anti-PTEN antibody, anti-VEGFA antibody, anti-PI3K p85 antibody, anti-PI3K p85 (phospho Y607) antibody, and anti-AKT1 (phospho S473) antibody were purchased from Abcam (batch no. Ab1; ab32199; ab1316; ab86714; ab182651; ab81283; dilution 1 : 200; 1 : 100; 1 : 100; 1 : 100; 1 : 200; 1 : 200); TRIB3 polyclonal antibody, AKT (L321) polyclonal antibody, and AKT (phospho-T308) polyclonal antibody were purchased from Bioworld (batch no. BS60451; BS1502; BS4647; dilution 1 : 100; 1 : 100; 1 : 100).