Af3242

The total protein from spinal cord tissues and cell samples was extracted using a protein extraction kit (Solarbio, Beijing, China), following the provided instructions, and the protein concentration was quantified using the BCA method. After gel preparation, the samples were loaded in equal amounts of total protein. The proteins was separated by 10% SDS-PAGE (80 V, 2 h, then 130 V, 1 h) and transferred to a PVDF membrane. The membrane was blocked with 5% milk for 1 h, followed by overnight incubation at 4°C with primary antibodies against NF200 (1:1000, 2,836, Cell Signalling Technology, Beverly, MA, United States), GAP43 (1:1000, 8,945, Cell Signalling Technology, Beverly, MA, United States), PI3K (1:1000, 4,292, Cell Signalling Technology, Beverly, MA, United States), p-PI3K (1:500, AF3242, Affinity, China), Akt (1:1000,bs-6951R, Bioss, Beijing, China), p-Akt (1:1000,bsm-60645R, Bioss, Beijing, China), and GAPDH (1:8000, 2,118, Cell Signalling Technology, Beverly, MA, United States). This was succeeded by incubation with HRP-conjugated secondary antibodies (1:10000, 7,074, 7,076, Cell Signalling Technology, Beverly, MA, United States) for 1 h at room temperature. Lastly, the membranes were visualized using ECL reagents and the Azure Biosystems NIR Fluorescence Imaging system, followed by quantitative grayscale analysis using Image J software.

Total protein was extracted from endometrium tissues using RIPA buffer (Servicebio, China) supplemented with 1% PMSF (Servicebio, China). Protein quantification was performed using an Enhanced BCA Protein Assay Kit (Servicebio, China). Sodium dodecyl sulfate polyacrylamide gel electrophoresis was carried out for protein separation. Then, the protein samples were transferred onto the PVDF membrane (Servicebio, China) and blocked in 5% BSA (Servicebio, China). Subsequently, the membranes were incubated with the primary antibodies against HOXA10 (ab191470, Abcam, UK), LIF (ab138002), VEGF (GB11034B, Servicebio, China), VEGFR2 (GB11190, Servicebio, China), P-PI3K (GB11190, Servicebio, China), AKT (GB111114, Servicebio, China), P-AKT (AF3242, Affinity, USA), and ACTIN (GB15001, Servicebio, China) at 4°C overnight. After washing with TBS-T three times, the membranes were then incubated with the secondary antibody at RT for 1h and detected using an ECL plus kit (G2019, Servicebio, China). PhotoShop software (alphaEaseFC, Alpha Innotech, USA) was used to remove the color, and Alpha software (Adobe PhotoShop, Adobe, USA) was used to analyze the optical density of the target band.

Total protein was extracted using RIPA lysis buffer containing phosphatase and protease inhibitors. Proteins were quantified using bicinchoninic acid (BCA) assay, and then separated with 10% SDS-PAGE gel and transferred to the PVDF membrane (Beyotime Institute of Biotechnology, Shanghai, China). The membrane was blocked with 5% BSA for 1 h and incubated overnight using rabbit antibodies to mTOR (1:1000, 2972S, Cell Signaling Technology), P-PI3K (1:1000, AF3242, Affinity Biosciences), P-mTOR (1:1000, 5536 T, Cell signaling Technology), PCNA (1:2000, 10205–2-AP, Proteintech Group), mouse antibodies to Actin (1:5000, 66009–1-Ig, Proteintech Group), PI3K (1:5000, 60225–1-Ig, Proteintech Group), AKT (1:2000, 60203–2-Ig, Proteintech Group), P-AKT (1:2000, 66444–1-Ig, Proteintech Group). Subsequently, HRP-conjugated secondary antibodies were treated, and the signal was measured using the enhanced chemiluminescence detection system (SH-523, Hangzhou, China). The intensity was analyzed by Image J software.

Western blot assay was performed as described before [26 (link)]. Briefly, total proteins of ESCs were extracted with RIPA (P0013, Beyotime, China) containing PMSF (ST506, Beyotime). After the measurement of protein concentration by the BCA kit (P0011, Beyotime), proteins were SDS-PAGE electrophoresed, transferred to PVDF membranes (IPVH00010, Millipore, USA), incubated for 1 h with 5% skim milk, and incubated at 4°C overnight with following antibodies: MMP-9 (1: 500, 10375-2-AP, Proteintech, China), FOXC1 (1: 500, A2924, ABclonal, China), PI3K (1: 1000, AF6241, Affinity, China), p-PI3K (1: 1000, AF3242, Affinity), p-Akt (1: 1000, AF6261, Affinity), Akt (1: 1000, AF0016, Affinity). After 4 times washing, the membrane was immune-blotted with secondary antibody for 40 min at 37℃. Protein expression was normalized to β-actin (1: 2000, 60008-1-Ig, Proteintech).

Total protein was extracted using radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) buffer containing phosphatase and protease inhibitors. The proteins were quantified using bicinchoninic acid (BCA) assay, separated via 10% SDS-PAGE and subsequently transferred to a polyvinylidene fluoride (PVDF) membrane (Beyotime Institute of Biotechnology, Shanghai, China). The membrane was blocked with 5% BSA for 1 h and then according to the molecular weight, the membrane was cut and incubated overnight with the corresponding primary antibody, including anti-Actin mouse antibody (66009–1-Ig, Proteintech Group, 1:5000), anti-PI3K mouse antibody (60225–1-Ig, Proteintech Group, 1:5000), anti-AKT mouse antibody (60203–2-Ig, Proteintech Group, 1:2000), anti-mTOR rabbit antibody (2972S, Cell Signaling Technology, 1:1000), anti-p-PI3K rabbit antibody (AF3242, Affinity Biosciences, 1:1000), anti-p-AKT mouse antibody (66444–1-Ig, Proteintech Group, 1:2000), anti-p-mTOR rabbit antibody (5536 T, Cell signaling Technology, 1:1000). After washing with Tris-buffered saline plus Tween®20 (TBST), the membranes were incubated with secondary antibody (1:5000) for 1 h, and chemiluminescence was used to visualize the protein bands with X-ray film. The intensity was analyzed by Image J software.

The OC tumor tissue and cells were extracted to obtain the protein, and the protein was quantified by using the BCA kit. The 50 μg protein was loaded quantitatively and separated by SDS-PAGE with 10% separation gel. Electrophoresis conditions were as follows: 80 V (30 min) and then 120 V; electrophoretic transfer condition was as follows: 90 min, 200 mA, and transfer protein to the PVDF membrane; it was then sealed for 2 h in skim milk powder solution; the corresponding primary antibodies anti-NF-κB p65 (ab16502, Abcam, Cambridge, 1 : 500), anti-p38 (Abcam, ab31828, 1 : 1000), anti-mTOR (phospho S2448)(Abcam, ab109268, 1 : 1000), phospho-PI3K p85 (AF3242, Affinity, 1 : 500), anti-AKT (phospho T308) (ab38449, Abcam, 1 : 500), anti-Bcl-2 (Affinity, AF6139, 1 : 1000), anti-Bax (Affinity, AF0120, 1 : 1000), anti-Cyt-C (Affinity, AF0146, 1 : 1000), anti-caspase-3 (Affinity, AF6311, 1 : 1000), anti-cleaved caspase-3 (Affinity, AF7022, 1 : 1000), and anti-GAPDH (ab8245, Abcam, 1 : 500) were added proportionally and incubated overnight at 4°C. After washing the membrane, a second antibody was added and incubated at room temperature for another 1 h. After washing the membrane again, the developer and fixative 1 : 1 were mixed; then, the protein strip was developed with a gel imaging system, and the statistical results were analyzed.