Ab463

Cardiac muscle (25–50 mg) was homogenized as previously reported [22 (link)] and 5μg of homogenate loaded for SDS-PAGE. Proteins were separated on a 6%, 7.5%, 10% or 12% resolving gel as required to optimize for MW separation, and transferred to polyvinylidene difluoride membrane (Roche, Laval, QC, CA). The following commercially available antibodies were used: total OXPHOS antibody cocktail (Abcam, Cambridge, MA, USA, ab110413, 1:500,), eNOS (Abcam, ab5589, 1:1000), VEGF (Abcam, ab46154, 1:1000), HIF1α (Abcam, ab463, 1:1000), alpha tubulin (Abcam, ab40742, 1:5000), muscle RING finger protein-1 (MuRF1; Santa Cruz Biotechnology, Dallas, TX, USA, sc-32920, 1:500), Muscle atrophy F-box (MAFbx; Santa Cruz, sc33782, 1:500), forkhead transcription factor-3a, Serine residue 253 (FOXO3a Ser253; Abcam, ab47285, 1:500), atrial natriuretic peptide (ANP; Abcam, ab180649, 1:500), BNP (Abcam, ab19645, 1:500) and beta-myosin heavy chain (β-MHC; Abcam, ab172967, 1:2000). All samples were detected from the same Western blot by cutting gels and transferring onto a single membrane to limit variability. Equal loading of protein was verified using Ponceau staining as well as constant alpha tubulin. All blots were quantified using enhanced chemiluminescence (Perkin Elmer, Woodbridge, ON, CA) and quantified by densitometry (Alpha Innotech Fluorchem HD2, Fisher Scientific, Ottawa, ON, CA).

Tumor tissues were dissected and stored at -80°C. The frozen tumor tissues were lysed with ice-cold RIPA Lysis Buffer (P0013B, Beyotime, China) for 30 minutes on ice then homogenized with a tissue homogenizer. The lysates were centrifuged at 12,000 rpm for 20 minutes at 4°C, and the protein concentration was determined by BCA Protein Assay (Beyotime, China). Equivalent samples (20 μg protein per lane) were subjected to SDS-PAGE on 10% gel. The proteins were then transferred onto polyvinylidene fluoride (PVDF) membranes (IPVH000 10, MercKMillipore), and incubated with primary antibodies against CA IX (ab38898, Abcam, USA, 1:1500), HIF-1a (ab463, Abcam, USA, 1:500) and a-tubulin (as internal reference, sc-80350, Santa Cruz, USA, 1:500) overnight at 4°C. Then membranes were incubated with anti-mouse or anti-rabbit secondary antibody for 1 hour at RT. Primary antibody was visualized by binding horseradish peroxidase (HRP)-conjugated secondary antibody with an Electro-Chemi-Luminescence (ECL) plus kit (Thermo).

Tissue were harvested and fixed with 4% formalin in PBS at 4°C for 24 h. The fixed samples were dehydrated, embedded with paraffin, and cut into 5-μm-thick sections. The sections were rehydrated, blocked with 5% goat serum, and incubated overnight at 4°C with the primary antibody of mouse anti-HIF-1α monoclonal antibody and rabbit anti-VEGF polyclonal antibody (1:200, Abcam, Hong Kong, ab463 and ab53465). The sections were then incubated with the secondary antibody. The labeled samples were then counterstained with 3–3'-diaminobenzidine.

Total protein was extracted from EC cells using buffer containing Halt Protease Inhibitor Cocktail (#87786, #78429, and #78438; Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein (30 μg) were separated by 10–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane, which was blocked with 5% non-fat milk and incubated overnight at 4°C with antibodies against DPPIV (1:400, ab28340), HIF-1α (1:500, ab463), VEGFA (1:400, ab51745) (Abcam) or β-tubulin (1:1000, #2128; Cell Signaling Technology, Danvers, MA, USA). Membranes were then incubated with a peroxidase-conjugated secondary antibody for 2 h at room temperature. Immunoreactivity was visualized by enhanced chemiluminescence using an Alpha Innotech Imaging System (Protein Simple, Santa Clara, CA, USA). Protein band intensity was normalized to the level of β-tubulin. Each experiment was repeated at least three times.

All animal procedures including uterine collections were performed in accordance with the guidelines of the Committee for Experimental Animals at Zennoh Embryo Transfer (ET) Center (Hokkaido, Japan) and the approval was also obtained from the Ethics Committee of the University of Tokyo (IRB number 7A-6-605). Paraffin sections of endometrial tissues were immunostained using antibodies targeting NFIL3, CEBPA and HIF1A, according to a previously described protocol (41 (link)). Briefly, the paraffin sections were rehydrated, boiled for 20 min in 10 mM citrate buffer (pH 6.0), and then incubated with an antibody against NFIL3 (1:100, Sigma-Aldrich, Saint Louis, MO, USA), CEBPA (1:100, ab15047, abcam), or HIF1A (1:100, ab463, abcam) overnight at 4°C. Subsequently, the paraffin sections were incubated with goat anti-rabbit IgG biotin conjugate (1:800 dilution, B8809, Sigma-Aldrich). The immunoreactivity was visualized by means of avidin-peroxidase (Sigma-Aldrich) and AEC substrate kit (Invitrogen) according to the manufacturer’s instructions.