Pvdf membranes 0.45 μm

Western blotting (WB) was performed to quantify the protein expression of hypoxia-inducible factor-2α (HIF-2α, Abcam, AB199) in bone marrow, and Caspase 3 (GeneTex, GTX110543) in blood. β-actin (ZS, TA-09) was performed as an internal reference protein. Briefly, blood cells and bone marrow cells were collected and lysed in radio immunoprecipitation assay (RIPA) buffer (Beyotime, China), and then Bicinchoninic Acid (BCA) protein assay (Thermo, USA) was used to determine protein concentration. 40 μg protein extracts were separated via polyacrylamide gel electrophoresis (PAGE) electrophoresis, and then transferred onto polyvinylidene fluoride (PVDF) membranes (0.45 μm, Millipore, USA). The membranes were blocked with 5% non-fat milk for 1 h under agitation, and then incubated with the primary antibody overnight. Subsequently, after washing 3 times with Tris Buffered Saline Tween (TBST), the membranes were incubated with the secondary antibody for 40 minutes at room temperature. Finally, enhanced chemiluminescence (ECL) detection reagents (Millipore, USA) were used to develop the protein, and Gel Image system ver.4.00 (Tanon, China) was carried out for quantifying the gray level, and β-actin bands were used for normalization.

After being treated with different concentrations of E5 (1–100 μM) for 24 h, cells were collected and subjected to caspase-3 signaling analysis. The western blot was performed as described previously.^{21 (link)} Briefly, cells were lysed in RIPA lysis buffer with protease and phosphatase inhibitors at 4 °C for 30 min. The lysates were centrifuged for 15 min at 12 000 r.p.m. The proteins were loaded and resolved on 12% polyacrylamide gels (Applygen, Beijing, China) and electroblotted to PVDF membranes (0.45 μm, Millipore, Bedford, MA, USA). The blots were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 2 h and probed with anti-cleaved caspase-3 antibody in 5% non-fat milk in TBST overnight at 4 °C. After being washed with TBST, the blots were incubated with the corresponding secondary antibodies. After a final wash, the antibody-antigen complexes on the blots were detected using Image Quant LAS 4000 (GE Healthcare Life Science, Pittsburgh, PA, USA).

The total protein was collected using trypsin with 0.25% ethylenediaminetetraacetic acid, PBS, and cell lysis buffer, and its quantity was measured by performing the bicinchoninic acid protein assay (40 (link)). The lysate (40 µg) was separated using SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (0.45 μm, Millipore, USA) by using a Trans-Blot TurboTM (Bio-Rad, USA). The membranes were blocked with 5% (w/v) bovine serum albumin (BSA) dissolved in Tris-buffered saline Triton-X100 (TBST), and each was incubated with a different primary polyclonal antibody, i.e., against TLR4 (1:1000), MyD88 (1:1000), TRAF6 (1:1000), IRAK1 (1:1000), IRAK4 (1:1000), NF-κB p65 (1:1000), p-NF-κB p65 (1:1000), IκB-α (1:1000), p-IκB-α (1:1000), COX-2 (1:1000), iNOS (1:1000), or GAPDH (1:20000), overnight at 4°C, and all dilutions were in TBST blocked with 5% BSA. The membranes were analyzed using ECL reagents.

Tissue samples were lysed in RIPA lysis buffer (#P0013B, Beyotime, Haimen, China) containing phenylmethane sulfonyl fluoride (PMSF) (#ST506, Beyotime, China). Subsequently, tissue samples were homogenized with a ball mill to obtain the total protein lysate. Protein concentrations of the tissue lysates were determined using a BCA protein assay kit (#P0010, Beyotime, China). Equal amounts of protein lysates were then separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (0.45 μm, Millipore, Darmstadt, Germany). PVDF membranes were blocked for 1 h at room temperature with Tris-buffered saline containing 0.05% Tween 20 and 5% powdered non-fat milk, and then incubated overnight with primary antibodies at 4 °C with shaking. Following three 5-min washes with Tris-buffered saline and 0.05% Tween 20, a secondary antibody was added and incubated for 1 h at room temperature. After washing (3 × 5 min), the membranes were visualized by chemiluminescence detection using Luminata Crescendo Western horseradish peroxidase (HRP) substrate (WBLUR0100, Millipore) according to the manufacturer’s instructions. Blots were imaged with the ChemiDoc XRS (Bio-Rad, Hercules, CA, USA) and analyzed using Image J software (http://imagej.nih.gov/ij/).

Total protein from sow endometria and mouse uteri was extracted as described above. Equal amounts of protein were loaded and separated on SDS-PAGE gels and then transferred to PVDF membranes (0.45 μm, Millipore, MA, USA). After blocking with 5% skim milk or bovine serum albumin for 1 h at room temperature, membranes were incubated with corresponding primary antibodies (Supplemental Table 2) overnight at 4°C. After five washes with TBST, membranes were incubated with DyLight 800-labeled secondary antibodies (Cell Signaling Technology, MA, USA) for 2 h at room temperature. Membranes were washed and then visualized with the Odyssey CLx (LI-COR, NE). Band densities were quantified using the ImageJ software. β-Actin was used as the loading control.