Ab170099

Cell lysate was collected using RIPA lysis buffer. The cell protein mass of each lysate was determined by BCA Protein Assay Reagent (Pierce, IL). Proteins were separated using 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to membranes (0.22 μm, Sigma) and incubated with primary antibodies at 4°C overnight. The primary antibodies are as follows: antibodies against VEGFA (1/1000; ab1316, Abcam), TGFβ1 (1/1000; ab215715, Abcam), Angiopoietin-1 (1/20000; ab183701, Abcam), ZO-1 (1/1000; ab216880, Abcam), Occludin (1/1000; ab216327, Abcam), Claudin-5 (1/1000; ab131259, Abcam), G3BP2 (1/2000; ab86135, Abcam), p-p38 (1/1000; ab195049, Abcam), p38 (1/2000; ab170099, Abcam), p-p53 (1/2000; ab33889, Abcam), p53 (1/1000; ab26, Abcam), and GAPDH (1/500; ab8245, Abcam). Next, the membranes were incubated with HRP-conjugated secondary antibody IgG (1/2000; ab7090, Abcam) at room temperature for 2 h. GAPDH antibody served as a negative control. At last, the protein bands were visualized by an ECL Western Blotting Substrate Kit (ab65623, Abcam) and quantified by the ImageJ software.

After the behavioral test, five mice randomly selected from each group were deeply anesthetized with 4% pentobarbital and then received a transcardiac perfusion of ice-cold sterilized saline (20 ml per mouse). The brain was taken out behind the ear, and then the hippocampus tissue was stripped and put into liquid nitrogen immediately, and finally preserved in the refrigerator at -80°C for use. After extracted and quantified, the supernatant of protein was denatured by boiling for 5 min in SDS sample buffer. 40 μg of total protein was separated by 6%–15% SDS-PAGE, blotted onto PVDF membranes, and then probed with the following antibodies: monoclonal amyloid-β antibody (sc-28365, Santa Cruz Biotech), monoclonal anti-Phospho-p38 MAPK antibody (#9216, Cell Signaling Technology), rabbit anti-Tau (phospho, S262) antibody (ab64193, Abcam), rabbit anti-Synaptophysin antibody (ab14692, Abcam), rabbit anti-p38 antibody (ab170099, Abcam), mouse anti-Tau antibody (ab80579, Abcam), and mouse anti-GAPDH antibody (BM3876, Bosterbio), conjugated to horseradish peroxidase were used as secondary antibodies. Protein bands were visualized by incubation with BeyoECL Plus (P0018, Beyotime, China) for 1 min and imaged by a Gel Image System (Tanon, 5200, China). Densitometry was performed by using ImageJ software.

Lung tissues (50 mg per mouse) were homogenized in ice‐cold RIPA buffer (Sigma‐Aldrich) with a 1% protease inhibitor cocktail and a phosphatase inhibitor, and protein concentration was examined using a BCA Protein Assay Kit (Beyotime, Shanghai, China). Thereafter, proteins (40 μg) were separated by 10% SDS‐PAGE and blotted onto a PVDF membrane. After blocking with 5% skimmed milk for 2 h, the membrane was incubated overnight with primary antibodies against p‐JAK2 (ab32101, 1:4000; Abcam), JAK2 (ab108596, 1:5000; Abcam), phosphorylated STAT3 (ab76315, 1:5000; Abcam), STAT3 (ab68153, 1:2000; Abcam), SOCS3 (ab280884, 1:1000; Abcam), p‐NF‐κB p65 (ab76302, 1:1000; Abcam), NF‐κB p65 (ab32536, 1:1000; Abcam), p‐p38 MAPK (ab195049, 1:1000; Abcam), p38 MAPK (ab170099, 1:1000; Abcam), and GAPDH (ab9485, 1:2500; Abcam) at 4°C, and subsequently incubated with secondary antibodies for 2 h at room temperature. The bands were visualized by enhanced chemiluminescence reagent (Beyotime), and the intensity of blot was quantified by Image Lab 3.0 software (Bio‐Rad, Hercules, CA, USA).

Total protein samples were extracted from penile tissues. The penile tissues were homogenized by precooled tissue lysates and centrifuged at 12,000 rpm for 15 min at 4°C, and the supernatants were obtained. Protein concentrations were determined using a BCA protein assay kit (Solarbio, Beijing, China). Protein lysate of equal concentration was loaded on 10% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) at 80 V for 30 min and 120 V for 60 min, and then electrotransferred to a polyvinylidene difluoride membrane (Bio-Rad, CA, U.S.A.). The membrane was blocked for 1 h at room temperature with 5% skimmed milk powder. Then, membranes were incubated with primary antibodies against CaSR (1:1000; ab18200, Abcam), PLC (1:5000; ab76155, Abcam), PKCδ (1:5000; ab182126, Abcam), JNK (1:2000; ab208035, Abcam), p38 (1:1000, ab170099, Abcam), Bax (1:2000, ab32503, Abcam), GAPDH (1:5000, 60004-1-1g, HuaBio, Hangzhou, China) at 4°C overnight, followed the membranes were washed and incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. Finally, the protein bands were visualized using an enhanced chemiluminescence detection system (Clinx Science Instruments, U.S.A.).