Chemiluminescent detection

Human neuronal cell line SK-N-SH were seeded at a density of 1.2×10⁵ cells/well in culture medium. To detect the expression of C99 and C83, the SK-N-SH cells were seeded at a density of 1×10⁶ cells/well. On the following day, the cells were treated with PL402 for another 24 hours followed by washing twice with PBS. The cells were then lysed with Laemmli sample buffer. The cell lysates were boiled and resolved by SDS/PAGE and then transferred to a nitrocellulose membrane. The interested proteins were recognized by corresponding primary antibodies and the blots were analyzed by the chemiluminescent detection (BioRad) of a peroxidase-conjugated, subtype-specific antibody (1:1000, Abmart). And the quantification for WB was used the image J software and normalized to actin.

RAW264.7 cells were lysed in RIPA buffer for 30 min on ice. After centrifugation at 12000 rpm for 15 min at 4 °C, protein concentration was measured in the supernatants by the BCA method. Next, 40 μg of protein were separated by SDS–PAGE and then transferred onto PVDF membranes (GE, USA). The membranes were blocked with 5% milk for 1 h and incubated with the primary antibody overnight at 4 °C. The membranes were washed three times with Tris Buffered Saline-Tween 20 (TBST), and then incubated with the corresponding secondary antibody for 1 h at room temperature. Finally, chemiluminescent detection (Bio-Rad, USA) was performed.

Aβ₄₀ and Aβ₄₂ in APP/PS1 mouse hippocampus and cortex were extracted as previously reported (Lazarov et al., 2005 (link)), for ELISA measurement of human Aβ₄₀ and Aβ₄₂, the frozen hippocampal and cortical tissue (400 mg) stored in −80°C were homogenized in 1 ml 2% SDS (dissolved in PBS), then centrifuged at 1,20,000 g for 60 min at room temperature. The supernatant was collected as the soluble fraction and quantified with human Aβ ELISA kits according to the users’ guidelines (ExCell Bio). Total Aβ levels in HEK293/APPswe cell culture medium were also quantified with ELISA.
Supernatant is also used for Western blot analyses. Proteins in supernatant were separated by SDS-PAGE, and transferred onto membrane. Proteins were labeled with β-actin (AB0035, Abways) and IL-1β rabbit polyclonal antibody (16806-1-AP, Proteintech) and the immunoreactive bands were detected by chemiluminescent detection (Bio-Rad) of peroxidase-conjugated antibody (M21002, Abmart). The intensity of each band was quantified by ImageJ and normalized to β-actin.