Ecl detecting system

Western blotting was performed on placental and whole-cell extracts. For placental samples, protein loading was normalized using Ponceau staining and densitometry. The aromatase (ab18995) rabbit polyclonal antibody (final dilution 1:2000) and hypoxia-inducible factor–1 alpha (HIF–1α) mouse monoclonal antibody (1A3) (final dilution 1:1000) were purchased from Abcam (Cambridge, USA), and the β-Tubulin (H–235) rabbit polyclonal antibody (final dilution 1:1000) was purchased from Santa Cruz Biotechnology (Santa Cruz, USA). Primary antibodies were detected using horseradish peroxidase-linked anti-rabbit or anti-mouse conjugates as appropriate (KPL, Gaithersburg, USA) and visualized using an ECL detecting system (Thermo Scientific, USA). The images derived from western blotting were analyzed by ImageJ (National Institutes of Health, Bethesda, MD, USA) software. Samples were normalized for protein loading by using β-Tubulin or Ponceau S staining.

Protein from cells or tissues was extracted using BCA kit. Protein was separated by 10% SDS–PAGE and then transferred onto PVDF membranes (Millipore Corporation, USA). The membrane was blocked with 5% milk and then incubated with primary antibodies (ROCK1, GAPDH, Cell Signaling Technology, USA). The membranes were incubated with the second antibodies and then measured using the ECL detecting system (Thermo Scientific, USA). GPADH was used as loading control.

Typical exosome markers were identified in the EV fraction by Western blot according to previously published methods. In brief, 10 μg of EVs proteins was separated on a 10% polyacrylamide gel. Separated proteins were transferred to polyvinylidene difluoride membrane (PVDF; Thermo Scientific) in transfer buffer for 1 h at 100 V. The membrane was washed in wash buffer (PBS TWEEN 20 (0.1%) three times for 10 min and blocked with 5% skimmed milk in PBS TWEEN 20 (0.1%) for 1 h at room temperature under agitation. The blocked membrane was probed for previously identified exosome-specific markers using the following primary antibodies: rabbit polyclonal anti-CD63 (1:1000, sc-15363, Santa Cruz Biotechnology), mouse monoclonal anti-Alix (1:1000, sc-53540, Santa Cruz Biotechnology), and rabbit polyclonal anti-Flotillin-1 (1:1000, 3253, Cell Signaling), diluted in 5% skim milk in PBS TWEEN 20 (0.1%) at 4 °C overnight on the laboratory rocker. After overnight incubation, the membrane was washed 3 times for 10 min in wash buffer. Bound antibodies were detected using horseradish peroxidase linked anti-rabbit or anti-mouse secondary antibodies conjugates (KPL, Gaithersburg) and visualized using an ECL detecting system (Thermo Scientific).