Ab56416

Mouse brain tissue sections and cells were incubated with following specific antibodies: anti‐LC3B (PM036, MBL), anti‐Iba‐1 (019‐19741, Wako), anti‐NeuN (MAB377, Millipore), anti‐P62 (ab56416, Abcam), anti‐LAMP2 (sc‐18822), anti‐ki67 (ab15580, Abcam), anti‐MAP2 (ab11267, Abcam), anti‐MBP (ab62631, Abcam), and anti‐Iba1 (019‐19741, Wako Pure Chemical Industries, OSA) in a humidified container for 12 h at 4°C, followed by incubation with fluorescent conjugated secondary antibodies (Santa Cruz Biotechnology).

The autophagy, proliferation, and apoptosis of CCECs after treatment were analyzed by western blot as described previously [36 (link)]. Briefly, all the CCECs were harvested from each lower chamber, and proteins were extracted using RIPA lysis buffer (Cwbiotech, China) containing proteinase inhibitor cocktail (Cwbiotech) and phosphatase inhibitor cocktail (Cwbiotech). The primary antibodies included anti-PCNA (1 : 1000; 200947-2E1, ZenBio, China), anti-cleaved-caspase3 (1 : 1000; 9661, Cell Signaling Technology, USA), anti-LC3 A/B (1 : 1000; 4108, Cell Signaling Technology), anti-p62 (1 : 1000; ab56416, Abcam), anti-Beclin1 (1 : 2000; ab207612, Abcam), and anti-GAPDH (1 : 2000; Affinity Biosciences, USA) antibodies. PCNA is an indicator of cell proliferation and cleaved-caspase3 is an indicator of cell apoptosis. LC3, p62, and Beclin1 are autophagic markers. GAPDH was used as loading control.

The corpus cavernosum tissues were lysed, and western blot analysis was performed as described previously [36 (link)]. The primary antibodies included anti-CD31 (1 : 2000; ab222783, Abcam), anti-eNOS (1 : 500; ab76198, Abcam), anti-phosphor-eNOS (S1177) (1 : 1000; 9571, Cell Signaling Technology), anti-VEGFRA (1 : 200; ab1316, Abcam), anti-VEGFR2 (1 : 1000; 9698, Cell Signaling Technology), anti-LC3 A/B (1 : 1000; 4108, Cell Signaling Technology), anti-p62 (1 : 1000; ab56416, Abcam), anti-Beclin1 (1 : 2000; ab207612, Abcam), and anti-GAPDH (1 : 2000; Affinity Biosciences, USA) antibodies. CD31, eNOS, phosphor-eNOS (S1177), VEGFRA, and VEGFR2 are indicators of endothelial function.

The PR-M(+) cells were lysed in a lysis buffer containing proteinase and phosphatase inhibitor at 4°C for 30 min and centrifuged at 10,000 r/min for 15 min. The cell lysates were then separated using 10% PAGE followed by transfer onto a polyvinylidene fluoride (PVDF) membrane. Membrane was blocked using 5% skim milk for 1 h at room temperature, followed by incubation at 4°C overnight with Tris-buffer saline with Tween (TBST)-diluted primary antibodies: anti-p62 (1:1000, ab56416, Abcam), anti-LC3B (1:10000, L7543, Sigma–Aldrich, St. Louis, MO, U.S.A.), anti-Bcl-2 (1:1000, sc-7382, Santa Cruz), anti-Beclin1 (1:500, sc-48341, Santa Cruz), anti-β-actin (1:5000, sc-47778, Santa Cruz) as an internal control, anti-PR (A-2) (1:5000, sc-398898, Santa Cruz), anti-PR-M (C262; 1:1000, sc-53943, Santa Cruz), anti-PR-B (B-30; 1:2000, sc-811, Santa Cruz) and anti-caspase-3 (1:1000, ab2302, Abcam). The membrane was then incubated with the horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h: goat anti-mouse IgG (1:5000, ab6789, Abcam) or anti-rabbit (1:5000, ab6721, Abcam). Finally, the membrane was washed six times with TSBT and developed using enhanced chemiluminescent (ECL), with the gray value of the protein bands quantified by ImageJ 1.48u software (Bio-Rad, Hercules, CA, U.S.A.). The experiments were repeated three times.

Podocytes were lysed using RIPA buffer, and protein concentration was determined using the BCA protein assay kit. Approximately 30 μg of protein from each sample was separated using a 10% SDS-polyacrylamide gel and transferred to PVDF membranes. Membranes were blocked with 5% skim milk in TBST and incubated with primary antibodies overnight at 4°C. Membranes were then incubated with the corresponding secondary antibodies for 1 h at room temperature and washed in TBST. Proteins were detected using specific antibodies: HMOX1: Abcam, ab13248; Sirt1: Abcam,ab110304; LC3B:Abcam,ab63817; AMPK:Abcam,ab110036; p-AMPK: Abcam,ab194920; P62: Abcam,ab56416; beta-actin: Abcam: ab115777.