Ab184238

The PVN tissue was lysed with RIPA buffer (P0013B, Beyotime, China) containing PMSF (ST506, Beyotime, China) on ice for 30 min. The protein samples were loaded onto 12% SDS-PAGE gels, electrophoresed, and transferred to PVDF membranes (Millipore Sigma, Burlington, MA, United States). The PVDF membranes were blocked with 5% (w/v) nonfat milk at room temperature for 2 h and then incubated overnight at 4°C with primary antibodies against CRH (ab184238, Abcam, Cambridge, United Kingdom) or β-tubulin (10094-1-AP, Proteintech, United States). The PVDF membrane was visualized by enhanced chemiluminescence (ECL kit, WBKLS0500, Millipore, Germany), and the protein concentration was quantified with the Quantity One software (version 4.0.3) from Bio-Rad (Hercules, CA, USA). After the lanes on the image were defined, the band densities of CRH and β-tubulin were measured separately. The density of the CRH protein band was normalized to the corresponding β-tubulin for each sample.

Western blots were performed using THP1 cells. Whole cell protein extracts were prepared using radioimmunoprecipitation assay (RIPA) buffer as reported earlier [17 (link)]. Protein concentrations were quantified using Bradford method and samples were prepared in sample loading buffer. Proteins were resolved on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels, transferred onto polyvinylidene fluoride (PVDF) membranes and probed with anti-UCN1 (PA5-75189, Invitrogen, CA, USA), anti-UCN2 (LS-C-335681-200, Lifespan Biosciences, Seattle, WA, USA), anti-UCN3 (bs-2786R, Bioss antibodies, Woburn, MA, USA), anti- CRF (ab184238, Abcam, Cambridge, UK), anti-SPEXIN (LS-C73416, Lifespan Biosciences, Seattle, WA, USA) and anti-GAPDH (ab2302, Millipore, Burlington, MA, USA) overnight at 4 °C. Following washes and incubation with rabbit horseradish peroxidase-conjugated secondary antibody, proteins were detected using West Femto ECL reagent (Thermo Scientific, Waltham, MA, USA). Protein bands were captured using Versadoc 5000 system (Bio-Rad, Hercules, CA, USA) and GAPDH was used as an internal loading control.

The hypothalamus, pituitary, and cavernous body tissues were individually ground, homogenized using RIPA lysis buffer. The homogenates were rested on ice for 30 min, and immediately subjected to centrifugation with 12,000 × g in 4°C for 15 min, then the supernatant was collected. The total protein concentration in supernatant was determined according to the BCA Protein Assay Kit (Cwbio). 50 µg of total protein was separated using SDS-PAGE. The electrophoresis peptides were transferred to the PVDF membranes. The PVDF membranes were then blocked for 2 h using 5% nonfat dried milk and probed with antibodies at 4°C. After washing 3 times using TBST, the PVDF membranes were incubated for 2 h using horseradish peroxidase-conjugated secondary antibodies, and then ECL (a chemiluminescence agent) was used to develop the protein bands. The signal intensity was analyzed using Gel-Pro Analyzer 4.0 software (Media Cybernetics, Silver spring, MD, United States). The GAPDH was used as an internal control to assess the protein loading equivalence. The eNOS antibodies (1:500,ab300071), PDE5A1 antibodies (1:500, ab259945), ACTH antibodies (1:500, ab32893), CRH antibodies (1:500, ab184238), and GAPDH antibodies were purchased from Abcam.