Hrp conjugated secondary antibody

SDS-PAGE gels (8–12%) were prepared to perform the Western blot assays. The proteins in gels were transferred on PVDF membranes using 200 mA for 90 min, followed by the PVDF membranes were blocked by 3% BSA. The proteins of PTPN14, ZEB1 and E-cadherin were detected with anti-PTPN14 mouse monoclonal antibody, anti-ZEB1 mouse monoclonal antibody and anti-E-cadherin rabbit antibody. GAPDH proteins served as an internal control. All antibodies were purchased from Cell Signaling Technology. The membranes were next incubated with HRP-conjugated secondary antibody (Zhongshan Biotechnology, China). The proteins were detected using SuperSignal® West Dura Extended Duration Substrate kit (Thermo).

Bovine GCs from each group were lysed with RIPA lysis buffer (Beyotime, Shanghai, China) having proteinase inhibitors. BCA Protein Assay Kit (Beyotime) was used for the determination of the extracted protein concentration. Equal amounts of protein (10 μg) were subjected to 10% SDS-polyacrylamide gel electrophoresis, and transferred onto a polyvinylidene difluoride (PVDF) membrane for 60 min. For blocking the membrane, skim milk in Tris-buffered saline (20 mM Tris-HCl, pH 7.6, 137 mM NaCl) with 0.1% Tween-20 (TBST) was used for 60 min at 38 °C and incubated with primary antibodies: anti CAT, BAX, Caspase-3, PCNA, CyclinB1, STAR, Cyp11A1, and β-actin (Cell Signaling Technology, Beverly, MA, USA), at 4 °C overnight. After washing with TBST thrice, membranes were incubated with HRP-conjugated secondary antibody (Zhongshan Biotechnology, Beijing, China) for 1 h at room temperature. The bands of protein were visualized through a chemiluminescence detection kit (Tanon, Shanghai, China). The bands were measured using the Image J 1.44p software and β-actin was used as a reference protein for normalization.

Formalin-fixed tissues from both ICP and control groups were embedded in paraffin, sectioned at 5 μm, and mounted on silane-coated slides. Sections were de-waxed and rehydrated by descending grades of alcohol and finally distilled water; endogenous peroxidase was blocked using 3% (v/v) hydrogen peroxidase in phosphate buffered saline (PBS). The sections were subjected to microwave antigen retrieval in 0.02 M EDTA, washed in PBS, and blocked with goat serum (Beijing ZhongShan Biotechnology) for 2 h. Sections were subsequently incubated overnight at 4°C with anti-ACOX1 (1:250). Sections were incubated with HRP-conjugated secondary antibody (1:1,000; Beijing ZhongShan Biotechnology) for 1 h at room temperature after being washed three times in PBS. Immunoreactivity was demonstrated using diaminobenzidine (Beijing ZhongShan Biotechnology) for increased sensitivity, which produced a brown insoluble precipitate at immunopositive sites. Sections were counterstained with hematoxylin and mounted with a cover glass. Negative controls were incubated with a solution devoid of primary antibodies. All immunostained sections were evaluated in a blinded manner by two observers. At the same time, Image J software was used to determine the intensity of staining in various parts of the placental sections from ICP and control groups.

Cells or tissues were lysed using RIPA lysis buffer containing protease inhibitor cocktail (Sigma). The protein concentration was determined using the bicinchonic acid protein assay kit (Applygen Technologies Inc., Beijing, China). The proteins (20 μg/lane) were separated on 10% SDS-PAGE gel and electrotransferred onto PVDF membranes (Millipore, Bedford, MA, USA). The membranes were blocked on Tris-buffered saline (pH 7.4) containing 5% non-fat milk at room temperature for 1 h and incubated with the primary antibodies overnight at 4 °C. The membranes were washed twice with appropriate HRP-conjugated secondary antibodies (Zhongshan Biotechnology Co.) at room temperature for 1 h. Antigen/antibody complexes were visualized using an enhanced chemiluminescence detection kit (Zhongshan Biotechnology Co.).

Immunohistochemical analyses were carried out as previously described [20 (link)]. The primary and secondary antibodies used in this study were SOX9, Peli2, and HRP-conjugated secondary antibodies (Zhongshan Biotechnology, Beijing, China). For each section, ten images were randomly captured at 200× magnification under a light microscope. The total cells and the SOX9- or Peli2-positive cells in each image were counted automatically using ImageJ software. After calculating the average of ten images, excluding the minimum and maximum values, the positive ratio of SOX9- or Peli2-expressing cells was determined; six sections per group of mice were taken for statistical analysis.

Salinomycin, doxorubicin and PKF 118-310 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Total-FOXO3a (t-FOXO3a), phospho-FOXO3a (Thr32; p-FOXO3a), total-AKT (t-AKT), phospho-AKT (Ser473; p-AKT), E-cadherin, Vimentin, β-catenin and TCF4 primary antibodies for western blotting, immunoprecipitation or immunofluorescence were obtained from Cell Signaling (Danvers, MA, USA). GAPDH primary antibodies were obtained from Kangchen Biotechnology (Sichuan, China). The HRP-conjugated secondary antibodies, goat-anti-mouse second antibody and goat-anti-rabbit second antibody, were purchased from Beijing ZhongShan Biotechnology Company (Beijing, China). Propidium iodide (PI) and anti-rabbit Alexa Fluor 488 (AF488) secondary antibody were both purchased from Invitrogen (Carlsbad, CA, USA).

After transfection for 48 hrs, the cells were first lysed with RIPA lysis buffer (Beyotime, Shanghai, China) containing a cocktail of protease inhibitors and phosphatase inhibitors for 30 min. on ice and were then centrifuged at 10,303 g for 20 min. The total protein concentration was measured by using a Bradford assay. Equal amounts of the protein samples were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and blotted onto a nitrocellulose membrane (Millipore, Bedford, MA, USA). The blot was blocked for 1 hr and then incubated overnight with a primary antibody against EGFR (Abcam, Cambridge, UK) and β-actin (Santa Cruz, CA, USA). After extensive rinsing, the blot was incubated with HRP-conjugated secondary antibodies (Zhongshan Biotechnology, Beijing, China) for 2 hrs at room temperature (RT). The immunoreactive bands were detected with an enhanced chemiluminescence reagent (Millipore, Billerica, MA, USA).