0.45 μm polyvinylidene fluoride membrane

The parasite proteins were extracted in reducing sample buffer for SDS-PAGE. Five micrograms of recombinant PvRALP1-Ecto or PvRALP1-Tr protein were loaded into each well and separated by SDS-PAGE under reducing conditions. The separated proteins were transferred to 0.45 μm polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) in a semidry transfer buffer (50 mM Tris, 190 mM glycine, 3.5 mM SDS, 20% methanol) at 400 mA for 40 min using a semidry blotting system (ATTO Corp., Tokyo, Japan). After blocking with 5% skim milk in phosphate-buffered saline containing 0.2% Tween 20 (PBS-T), the membranes were probed with mouse anti-PvRALP1-Ecto and anti-PvRALP1-Tr sera, rabbit anti-PvRALP1-Tr serum, anti-GST monoclonal antibody (Novagen, Madison, WI, USA), anti-penta-His monoclonal antibody (Qiagen), preimmune mouse serum, pooled sera from P. vivax malaria patients or noninfected individuals, all diluted 1:200 in PBS-T. IRDye goat anti-mouse, IRDye goat anti-rabbit, or IRDye goat anti-human sera (LI-COR Biosciences, Lincoln, NE, USA) were used to detect recombinant proteins according to the manufacturer’s instructions. Data were scanned with an Odyssey infrared imaging system (LI-COR Biosciences) and analysed with Odyssey software (LI-COR Biosciences).

Cells were lysed using radioimmunoprecipitation buffer (Beyotime, Shanghai, China). Total protein (20 μg) was separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto 0.45 μm polyvinylidene fluoride membranes (Millipore, Billerica, USA) using a Mini-Protean System (Bio-Rad, Hercules, USA). Membranes were incubated with primary antibody (1:500 dilution, catalog no. ABS1508, anti-SREBP1 antibody from Sigma-Aldrich; 1:500 dilution, catalog no. 3189S, anti-FASN antibody from Cell Signaling, Danvers, USA; 1:1 200 dilution, catalog no. AA128, anti-ACTB antibody from Beyotime) overnight at 4°C, then incubated with horseradish peroxidase-conjugated anti-rabbit antibody from Beyotime (1:8 000 dilution, catalog no. A0208) or anti-mouse antibody from Beyotime (1:12,000 dilution, catalog no. A0216) for 2 h at 37°C. Enhanced chemiluminescence (Beyotime) was added to the membranes, which were detected using an Odyssey Fc Imaging System (Li-Cor, Lincoln, USA) [22 (link)].

After cells or bone samples had been lysed, the protein concentrations were measured using a bicinchoninic acid protein assay kit. The lysates were centrifuged and denatured for 5 min at 94°C. Each protein sample underwent 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and was transferred onto 0.45-μm polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with Tween 20 (TBST) for 1 h, and probed with primary antibodies against RANKL (1:1000, R&D Systems), bcl-2 (1:1000, Abcam), WNT5A (1:1000, Abcam), or β-actin (1:5000, Abcam) at 4°C overnight. The membranes were then washed three times with TBST and incubated for 1 h with horseradish peroxidase–conjugated secondary antibodies (1:5000, Abcam). The proteins were visualized using an enhanced chemiluminescence detection system as recommended by the manufacturer.

Cells were lysed in radioimmunoprecipitation assay buffer containing protease inhibitors and were quantified using a bicinchoninic acid kit (Thermo Fisher Scientific, Rockford, IL, USA). Equal amounts of protein were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred to 0.45-μm polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked using Tris-buffered saline (TBS) and 0.1% Tween 20 (TBST) containing 5% bovine serum albumin for 2 h and then incubated with primary antibodies (anti-p53, -USP9X, and -GAPDH, diluted 1:1000 in TBST) overnight. After washing three times with TBST, the membranes were incubated with the appropriate secondary antibodies conjugated to horseradish peroxidase for 1 h at room temperature. Bands were visualized using enhanced chemiluminescence in the western blot detection system. All antibodies were purchased from Abcam (Cambridge, MA, USA).

Primary neurons were seeded in poly-D-lysine-coated 6-well plates at a density of 8×10⁵ cells/well. At DIV7, cells were treated with either Aβ42 species alone or together with inhibitors, as indicated in each experiment. After 24 h, the cells were harvested and lysed using radioimmunoprecipitation assay buffer (RIPA buffer) in the presence of protease and phosphatase inhibitor cocktails (Transgene, Beijing, China). The protein concentration was determined using a BCA protein assay kit (Beyotime). Protein samples (10-15 μg) were separated by SDS-PAGE and transferred to 0.45-μm polyvinylidene fluoride membranes (Millipore, Massachusetts, United States). Following transfer, the membranes were blocked with 5% BSA-TBST or 5% milk-TBST at room temperature for 1 h. The membranes were probed with primary antibodies and HRP-conjugated secondary antibodies. Target proteins were further detected using ECL chemiluminescent HRP substrate (Millipore) in a Bio-Rad Chemidoc imaging system (California, United States). All blots were analyzed using the ImageJ software (version 1.50i) to determine the optical densities. Data were normalized to the corresponding β-actin bands. The primary antibodies used in the study can be found in the Supplementary Information (Table S1).