Pgrmc1

Paraffin-embedded rat ovary tissues were prepared, followed by deparaffinization and rehydration. After antigen exposed, tissue slides were incubated with 3% H₂O₂ for 10 min and goat serum for 30 min to block non-specific binding. Primary antibodies: ANP (1:100; Abcam, Cambridge, UK), NPRA (1:100; Santa Cruz Biotechnology), NPRC (1:100; Santa Cruz Biotechnology), PGRMC1 (1:50; Cell Signaling), PGRMC2 (1:50; Cell Signaling), PCNA (1:200; Proteintech), p-c-Fos (1:50; Cell Signaling), c-Fos (1:50; Cell Signaling), p-c-Jun (1:50; Cell Signaling) and c-Jun (1:50; Cell Signaling) were incubated overnight at 4 °C. The second antibody was incubated for 40 min after washing with PBS. The signals were visualized with DAB wherein yellowish-brown stain indicated as a positive result. The negative control was obtained by replacing primary antibody with PBS. Images were taken under an inverted microscope.

Liver and Hep3B cells were lysed and sonicated for protein extraction. Protein samples were subjected to 8–12% SDS-PAGE after 5 min of boiling at 100 °C. Gels were blotted by wet transfer with Bio-Rad Power Pac at 350 V for 1 to 2 h. PVDF membranes were blocked for 1 h in bovine serum albumin (BSA) and incubated with primary antibodies overnight at 4 °C. Membranes were then incubated with secondary antibodies diluted with 1:10,000 in BSA overnight at 4 °C. The results were detected with enhanced chemiluminescence (ECL) solution (Cyanagen) and ChemiDoc (Fusion Solo, Vilber Lourmat). Primary polyclonal antibodies used were: rabbit anti-β-actin (sc-130656, Santa Cruz Biotechnology, USA) and rabbit anti-PEPCK (10004943, Cayman Chemical, USA). Rabbit monoclonal antibody to PGRMC1 (#13856) was purchased from Cell Signaling Technology, Inc. Rabbit monoclonal antibody to phosphor CREB (ab32096, Abcam), CREB (ab32515, Abcam) were used. The secondary antibody used was mouse anti-rabbit IgG (211-032-171 anti-rabbit, Jackson laboratory).

Protein samples were extracted with lysis buffer. 15 μg protein samples from each group were loaded onto the SDS polyacrylamide gel for electrophoresis and transferred to NC membranes (Millipore, Billerica, MA, USA). After blocking in 5% non-fat milk at room temperature for 2 h, primary antibodies: NPRA (1:1000; Santa Cruz Biotechnology), NPRC (1:1000; Santa Cruz Biotechnology), PGRMC1 (1:1000; Cell Signaling), PGRMC2 (1:1000), Bax (1:2000; Proteintech), Bcl-2 (1:2000; Proteintech), Caspase 8 (1:1000; Proteintech), Caspase 9 (1:1000; Proteintech), PCNA (1:2000; Proteintech), EGFR (1:500; Proteintech), p-EGFR (1:500; Cell Signaling), ERK 1/2 (1:500; Beyotime Biotechnology), p-ERK 1/2 (1:500; Beyotime Biotechnology), p-c-Fos (1:500; Cell Signaling), c-Fos (1:500; Cell Signaling), p-c-Jun (1:500; Cell Signaling), c-Jun (1:500; Cell Signaling) and GAPDH (1:2000; Proteintech) were incubated at 4 °C overnight. Next day, secondary antibodies (1:2000; Cell Signaling) were incubated for 45 min at room temperature. ECL detection system was used to visualize the bands. All experiments were repeated at least three separate times.