Amersham imager 800

4 μL of prepared cell or animal sample protein solutions were dotted for adsorption onto methanol-pre-activated PVDF membranes and then blocked with 5% non-fat dry milk in Tris-buffered saline and Tween 20 for 1 h. The samples were incubated overnight at 4 °C with anti-amyloid fibril primary antibody [mOC87] (Abcam, MA, USA), anti-amyloid oligomer A11 (Invitrogen, CA, USA) or purified anti-β-amyloid (1–16) antibody (clone 6E10) (Biolegend, CA, USA) (1:1000), followed by HRP-conjugated secondary antibody. Protein bands were detected using Super Signal Sensitive ECL Western Blotting Detection Reagent (Beyotime, Beijing, China) and observed using a gel imaging device (Amersham Imager 800, GE, Tokyo, Japan).

Infected macrophages were centrifuged, washed with PBS, and disrupted in lysis buffer (Keygen, KGP10100, Nanjing, China) in an ice-bath for 30 min. The lysates were centrifuged at 12,000× g, 4 °C for 15 min and supernatants were collected. Protein concentrations were determined by a BCA kit (Keygen, KGP903, Nanjing, China). SDS loading buffer was added, and samples were boiled at 100 °C for 5 min. Protein samples were separated by 12% SDS-PAGE and blotted onto PVDF membranes. The membranes were blocked with 10% nonfat powdered milk for 2 h and exposed to the relevant primary antibody for 24 h at 4 °C: rabbit anti-cleaved caspase-3 (1:1000; catalog number 9661; CST, Danvers, MA, USA), rabbit anti-LC3B (1:1000; catalog number ab192890; Abcam, Cambridge, UK), rabbit anti-P62 (1:1000; catalog number ab109012; Abcam), mouse anti-β-actin (1:5000; catalog number ab8226; Abcam). After washing, the blots were incubated with HRP-conjugated secondary antibody at RT for 2 h. The signal was visualized using an ECL chemiluminescence kit (Beyotime, P0018FS, Shanghai, China), and imaged by a gel imaging system (GE Amersham Imager 800, Boston, NY, USA).

Proteins were extracted from cells on ice using RIPA buffer (P0013B, Beyotime) containing a protease/phosphatase inhibitor cocktail (Roche), separated by SDS-PAGE, and transferred to PVDF membranes. The membranes were blocked in 5% nonfat dry milk/Tween 20-tris buffered saline (TBST) for 1 h and incubated overnight at 4 °C with corresponding primary antibodies as follows: anti-LKB1 (1:1000, #3047, Cell Signaling Technology), anti-LOX-1 (1:1000, ab60178, Abcam), anti-SR-A1 (1:1000, ab151707, Abcam), anti-CD36 (1:1000, ab133625, Abcam), anti-SR-B1 (1:2000, ab52629, Abcam), anti-ABCA1(1:1000, #96292, Cell Signaling Technology), anti-ABCG1 (1:1000, ab52617, Abcam), anti-SIRT6 (1:1000, #12486, Cell Signaling Technology), anti-p-AMPKα (1:1000, #2535, Cell Signaling Technology), anti-AMPKα (1:1000, #5831, Cell Signaling Technology), anti-Phospho-(Ser/Thr) (1:1000, ab117253, Abcam), anti-GAPDH (1:1000, #5174, Cell Signaling Technology), and anti-Tubulin (1:1000, 11224-1-AP, Proteintech). After three washes with TBST, the membranes were incubated with appropriate horseradish peroxidase (HRP) conjugated secondary antibodies. After washing, the membranes were added dropwise to immobilized ECL ultra-western HRP substrate (Millipore) and imaged using a luminescent image analyzer (Amersham Imager 800, GE).