Sc 19992

Brain tissue sections were rinsed in PBS and permeabilized with 0.3% Triton X-100 in PBS for 10 min at room temperature. After blocking with 10% normal goat serum for 1 h, sections were incubated overnight at 4°C with rabbit anti-NeuN (1:500, ab128886; Abcam), mouse anti-LC3 (1:100, M152–3; MBL), and rat anti-lysosomal-associated membrane protein (LAMP)1 (1:50, sc-19,992; Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies, followed by Alexa Fluor 647-, 594-, or 488-conjugated secondary antibody (1:500; Thermo Fisher Scientific; Massachusetts; USA) for 1 h at room temperature. Cell nuclei were visualized by counterstaining with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma–Aldrich). Sections were mounted on slides with 70% glycerol and covered with coverslips, and were visualized on a confocal microscope (TCS SP8; Leica, Solms, Germany). Five sections (distance between each section was 1mm) in hippocampus head were chosen from each monkey, and six cells in hippocampus CA1 or CA3 were randomly selected in each section. A total of thirty cells in hippocampal CA1 or CA3 were performed for evaluation in each monkey. Immunofluorescent intensity was measured via the ratio of cell expression to background in each view.

Formalin-fixed, paraffin-embedded tissue sections were incubated overnight at 4°C with rat anti-LAMP1 (SC-19992, Santa Cruz Biotechnology, 1:100), rat anti-LAMP2 (ab13524, Abcam, 1:500), rabbit anti-LIMP2 (NB400; Novus Biologicals, 1:100), rabbit anti-GFAP (Z0334, Dako Cytomation, 1:1000) and BSI-B4 lectin (L5391, Sigma, 1:100). For bright-field immunostaining, biotinylated rabbit anti-rat IgG (E0467, Dako, 1:300) and biotinylated goat anti-rabbit IgG (31820, Vector Laboratories, 1:300) were used as secondary antibodies. Bright-field sections were stained with 3,3-diaminobenzidine (Sigma) and counterstained with haematoxylin. Images were obtained with an Eclipse 90i optical microscope (Nikon). LIMP2, LAMP2, GFAP and BSI-B4 signals were quantified with the NIS-Elements Advanced Research 2.20 software (Nikon) in 3-5 20× images of each brain region of each animal using the same signal threshold settings for all images. The percentage positive area of each image was then calculated.

For immunoblotting and immunofluorescence, the following antibodies were used in this study: anti-FLAG (mouse; Sigma-AldrichF1804; Sigma-Aldrich), anti-α-tubulin (mouse; B512; Sigma-Aldrich), anti-TBK1 (rabbit; EP611Y; Abcam), anti-p-TBK1 (Ser172; rabbit; D52C2; Cell Signaling Technology), anti-LAMP1 (mouse; sc-19992; Santa Cruz Biotechnology), anti-p62 (rabbit; PM045; MBL), anti-ubiquitin antibody, Lys48-specific (rabbit; 05-1307; Millipore), anti-ubiquitin antibody, Lys63-specific (rabbit; 05-1308; Millipore), and anti-polyubiquitin antibody FK2 (mouse; 0918-2; Nippon Bio-Test Laboratories). The secondary antibodies used for immunoblotting were horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (111-035-003; Jackson ImmunoResearch) and HRP-conjugated goat anti-mouse IgG (115-035-003; Jackson ImmunoResearch). The secondary antibodies used for immunofluorescence were goat anti-rabbit Alexa Fluor 488 preabsorbed (ab1500085; Abcam) and goat anti-mouse IgG (H+L) cross-adsorbed Alexa Fluor 568 (A11004; Invitrogen). LLOMe was purchased from Sigma-Aldrich. Depending on the experiment, cells were treated for 1 h with 10 µM BAPTA-AM (Dojindo) or for 20 h with 250 nM bafilomycin A1 (Cayman Chemical).

Antibodies against p70S6K (sc-8418), LAMP1 (sc-19992), and phospho-mTOR (sc-293133) were purchased from Santa Cruz Biotechnology (USA). Antibodies against phosphor-p70S6K (AP0564), RRAGA (A15134), RRAGD (A9979), and ADORA1 (A5219), ADORA2A (A1587) were purchased from ABclonal (China). Antibodies against NT5C2 (15223-1-AP), RRAGB (13023-1-AP), RRAGC (26989-1-AP), SP1 (21962-1-AP), YY1 (66281-1-lg), mTOR (66888-1-Ig), Actin (66009-1-lg), GAPDH (10494-1-AP), ADA1 (13328-1-AP), MDH2 (15462-1-AP), MDH1 (15904-1-AP), ACO1 (12406-1-AP), CS (16131-1-AP), GLUD1 (14299-1-AP), and SDHB (10620-1-AP) were purchased from Proteintech (China). Antibody against PNP (DF8260) was purchased from Affinity (China). Antibody against puromycin (MABE343) was purchased from Sigma-Aldrich (USA). Antibody against Ki67 (ab15580) was purchased from Abcam (UK).

For confocal microscopy, we used the rat monoclonal anti-LAMP1 (sc-19992, Santa Cruz, 1:200). For WB, the following antibodies were used: mouse monoclonal anti-NCT (Esselens et al., 2004 (link)) (9C3, 1:7000), rabbit polyclonal anti-PSEN1-NTF (ab71181, Abcam, 1:2000) or rat polyclonal anti-PSEN1-NTF for co-immunoprecipitation (MAB1563, Millipore, 1:4000), rabbit monoclonal anti-PSEN1-CTF (ab76083, Abcam, 1:2000), rabbit polyclonal anti-PEN2 (ab18189, Abcam, 1:1000), rabbit polyclonal anti-APP-CTF (Esselens et al., 2004 (link)) (B63, 1:10,000) and mouse monoclonal anti-transferrin receptor (136800, Invitrogen, 1:4000). Secondary antibodies included HRP-conjugated goat-anti-mouse and goat-anti-rabbit (Bio-Rad, 1:10000 dilution). Co-immunoprecipitation was done with rabbit anti-GFP (#A11122; Invitrogen). For quantitative WB, mouse monoclonal anti-PSEN1-CTF (MAB5232, Millipore, 1:1000 dilution) and near-infrared goat anti-mouse Alexa Fluor790 (#A11375, Invitrogen, 1:15,000 dilution) were used. For PM sheet immunofluorescence, GFPbooster-Atto647N (Chromotek, 1:1000 dilution) was used.

For mouse immunofluorescence experiments, 5 μm thick frozen tissue sections were incubated with rat anti-LAMP1 antibody (sc-19992, Santa Cruz) or mouse anti-p62 antibody (ab56416, Abcam) for 15 hours after 60 minutes blocking with 5% BSA in 0.4% Triton X-100/PBS. Sections were then incubated with anti-rat IgG Alexa fluor 633 (A-21094, Thermo Fisher Scientific) or anti-mouse IgG2a CF405S (SAB4600476, Sigma) for 60 minutes. Slides were mounted with Vectashield Antifade Mounting Medium (H-1000, Vector) after nuclear counter staining with Hoechst 33342 (62249, Thermo Fisher Scientific) or DRAQ5 (ab108410, Abcam). Confocal images were acquired using a laser-scanning confocal microscope (LSM880, Zeiss). For phospho-S6 immunohistochemistry, 12 μm thick paraffin embedded sections were incubated with rabbit anti-phospho-S6 antibody (4858, Cell Signaling Technology) for 15 hours after citric acid antigen retrieval in a microwave for 2 minutes, H₂O₂ treatment, avidin-biotin blocking and 30 minutes blocking with 2% horse serum/TBS. Detection was done with VECTASTAIN Elite ABC-Peroxidase Kit (PK-6101, Vector) according to the manufacturer’s instructions followed by chromogen DAB staining, nuclear counter staining with hematoxylin and imaging by light microscopy (Axioplan 2 Universal Microscope, Zeiss)