Nci4106

Synchronized L1 worms were cultured for 36 h at 16°C on 100 mm NGM plates containing OP50 and drugs, upshifted to 23°C, and incubated at that temperature for another 32 h. The worms were then collected in 1x phosphate-buffered saline (PBS) (8 g NaCl, 0.2 g KCl, 1.44 g Na₂HPO₄, and 0.24 g KH₂PO₄). The total protein was isolated with ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer containing 1x protease inhibitor and 1x phosphatase inhibitor cocktail and identified by using a Tris-Tricine gel (each lane was loaded 40 μg protein) [11 (link)]. The Aβ protein levels were detected with 6E10 monoclonal antibody (1 : 500; BioLegend). Species-specific β-actin was the internal control detected with mouse β-actin monoclonal antibody (60008-1-Ig; 1 : 2000; Proteintech®). Anti-mouse IgG HRP-linked antibody was the secondary antibody (No. 7076, 1 : 3000; Cell Signaling Technology). Blots were visualized by standard enhanced chemiluminescence (ECL; NCI4106; Thermo Fisher). The protein signals were quantified with Gel-Pro Analyzer 4.

Cells were lysed using a radioimmunoprecipitation assay buffer, containing protease and phosphatase inhibitors. Proteins (60 μg) were separated using 10% discontinuous sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% non-fat milk for 1 h and then incubated with primary antibodies at 1:1000 dilution overnight at 4 °C. The membrane was washed in Tris-buffered saline containing 0.1% Tween 20 and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h. Protein bands were visualized by a densitometer using an enhanced chemiluminescence (ECL) detection kit (NCI4106; Thermo Fisher Scientific, Inc.) [37 (link)]. The same membrane was used for re-probing with an anti-GAPDH antibody (Cell Signaling Technology, Inc., Beverly, MA, USA) at 1:1500 dilution. Band intensities were estimated using Image J, and the expression of proteins was normalized with respect to that of GAPDH.
Antibodies used have been listed in Table 1.

Cells or tissue samples were lysed in RIPA lysis buffer (Tian P. et al., 2017 (link)) added protease inhibitor (P8340, Sigma, St. Louis, MO, United States) for 30 min on ice, and then centrifuged at 12, 000 rpm for 15 min at 4°C. The supernatant was collected and measured to calculate protein concentration using a BCA protein assay kit (23225, Thermo Fisher Scientific, Waltham, PA, United States). After denaturation, 80 μg protein was electrophoresed in a 10% SDS-PAGE, and then transferred onto a nitrocellulose membrane. The membrane was blocked with 5% skimmed milk powder in TBST (Tris buffer with 0.1% Tween, pH 7.6) for 2 h and then incubated at 4°C overnight with primary antibodies: DMT1 (ab55735, Abcam, 1:500) or β-actin (BS6007M, Bioworld, 1:10000). A secondary HRP-conjugated antibody (BS12478, Bioworld, 1:10000) was used to incubate the membrane for 2 h prior to chemiluminescent detection. The signals were determined using a chemiluminescent substrate (ECL) kit (NCI4106, Thermo Fisher Scientific, Waltham, PA, United States). Then, protein intensities were quantified by the VersaDoc MP 4000 system (Bio-Rad, California, CA, United States).