Anti uch l1

Proteins were resolved on 8%, 10%, 15%, or 4-20% tris-glycine gels, and for amyloid-β or Ub immunoblotting 16% tris-tricine gel was used^{62 (link)}, transferred to nitrocellulose membranes (GE Whatman, 0.2 µm Pore Size). Western blot images were visualized by enhanced chemiluminescence (ECL). The images were captured by using an ImageQuant™ LAS 4000 imaging system (GE Healthcare) and quantitated by Image Studio™ Lite (LI-COR Biosciences). Primary antibodies were used at the following dilutions: 6E10 anti-amyloid-β (1:1000, Biolegend, Cat. no. 803015); anti-PSEN1 (1:1,000, Cell Signaling Technology, Cat. no. 5643); anti-β-tubulin (1:1,000, Abcam, Cat. no. AB24629); anti-UBB⁺¹ (1:1000, custom made by Sigma for our lab), anti-ubiquitin (1:2000, DAKO, Cat. no. Z0458), anti-UCH-L1 (1:1000, Abcam, Cat. no. ab8189), anti-GAPDH (1:10,000, Sigma, Cat. no. G9545), anti-β-actin (1:10,000, Santa Cruz, Cat. no. sc-47778).

Human testis paraffin sections were routinely deparaffinized and hydrated. After antigen retrieval and endogenous peroxidase blocking, the sections were blocked with 5% BSA (Sigma-Aldrich, St. Louis, MO, USA) for 1 hour at room temperature and incubated with primary antibodies, including anti-FOXP3 (Abcam, catalogue: ab22510), anti-UCHL1 (Abcam), anti-PCNA (Abcam), and anti-Ki67 (Abcam), at 4°C overnight. Antibodies binding were detected with HRP conjugated second antibodies, stained by DAB (Vector Lab, Burlingame, USA) and counterstained with hematoxylin, or detected by Alexa Fluor-conjugated second antibody (Invitrogen, USA) (1:200, room temperature, 1 h). DAPI was utilized for staining cell nuclei. The images were captured using the fluorescence microscope (Nikon, Tokyo, Japan).