Goat anti rabbit hrp conjugate

hGCs and ovaries were gathered and dissolved in lysis buffer as previously discussed. Total protein was extracted and loaded on 10% gels. Then, the protein was separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), and the proteins were then electrotransferred to polyvinylidene difluoride membranes (PVDF, Millipore, USA). The membranes were incubated overnight at 4°C with primary antibodies. Then, appropriate secondary antibodies (goat anti-rabbit HRP conjugates; Jackson Immuno Research, West Grove, USA) were incubated separately with the membranes. After washing three times with Tris-buffered saline, a chemiluminescence kit was used to enhance specific signals (Pierce ECL Western Blotting Substrate, Thermo). Finally, the membrane was examined with an imaging detection system (Tanon, China), and ImageJ software (National Institutes of Health, USA) was used to analyze immunoreactive bands. All experiments were repeated at least three times. The results are presented as the mean ± standard deviation, and p < 0.05 was considered statistically significant. Information regarding the primary antibodies used for western blot analyses is listed in Supplementary Table 3.

Samples from differentiation days 14 and 30 were harvested and dissociated in a lysis buffer. Protein was extracted from each sample, which was then loaded on 10% gels and separated by SDS‐PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis). Next, the separated proteins were transferred to polyvinylidene difluoride membrane (PVDF, Millipore, USA). Thirdly, the proteins were incubated with the primary antibodies (Abcam, Cambridge, MA, USA) of anti‐human‐NESTIN, anti‐human‐TUJ‐1, anti‐human‐tyrosine hydroxylase (TH), anti‐human‐MSI‐1, anti‐human‐Gapdh, anti‐human‐β‐tubulin, anti‐human ERα, anti‐human ERβ and appropriate secondary antibodies (goat anti‐rabbit HRP conjugates; Jackson Immunoresearch, West Grove, PA, USA) separately. The specific signals were detected by the enhanced chemiluminescence (Pierce ECL Western blotting Substrate; Thermo). Finally, the membrane was checked by a chemiluminescence detection system (Tanon, Shanghai, China) and the signal intensity of each band was analysed by Imaging J Software (National Institutes of Health, USA). Experiments were repeated three times, results are presented as fold change ±SD and P < 0.05 is determined significant difference (The assay of used antibodies are shown in Table 2).

hAMSCs and hAECs from three pregnant women were harvested and dissociated in a lysis buffer. Protein was extracted from each sample, which was then loaded onto 10% gels and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Next, the separated proteins were transferred to polyvinylidene difluoride membrane (PVDF; Millipore, USA). Thirdly, the proteins were incubated with the primary antibodies (Abcam, USA) of anti-human-collagen I, II, III, IV, and VI, anti-human-telomerase reverse transcriptase, anti-human-OCT4, anti-human-NANOG, anti-human-Gapdh, anti-human-β-Tubulin, anti-human-SSEA4, or anti-human-TRA-1-81 and appropriate secondary antibodies (goat anti-rabbit HRP conjugates; Jackson Immunoresearch, West Grove, PA, USA) separately. The specific signals were detected by enhanced chemiluminescence (Pierce ECL Western blotting Substrate; Thermo). Finally, the membrane was checked by a chemiluminescence detection system (Tanon, China) and the signal intensity of each band was analyzed using Imaging J Software (National Institutes of Health, USA). Experiments were repeated three times, results are presented as fold change ± SD, and p < 0.05 is determined as significant difference.

hGCs from the POI patients, ovarian tissues from the POI mouse model, hGCs treated with PBS, exosomes, or hADSCs were harvested and dissociated in a lysis buffer. Protein was extracted from each sample, which was then loaded onto 10% gels and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Next, the separated proteins were transferred to polyvinylidene difluoride membranes (PVDF; Millipore, USA). The proteins were then incubated with the primary antibodies (Abcam, USA), namely, anti-human-SMAD2, anti-human-SMAD3, anti-human-SMAD4, anti-human-Fas, anti-human-FasL, anti-human-Caspase-3, anti-human-Caspase-8, anti-human-CD9, anti-human-CD63, anti-human-CD81, and anti-human-GAPDH, and subsequently incubated with the appropriate secondary antibodies (goat anti-rabbit HRP conjugates; Jackson Immunoresearch, West Grove, PA, USA). The specific signals were detected with enhanced chemiluminescence (Pierce ECL Western blotting Substrate; Thermo). Finally, the membrane was visualized with a chemiluminescence detection system (Tanon, China), and the signal intensity of each band was analyzed with ImageJ software (National Institutes of Health, USA). Experiments were repeated three times. The results are presented as the fold change ± SD. p < 0.05 is considered a statistically significant difference.