Ez capture mg system

The mouse hippocampi were lysed with radioimmunoprecipitation assay buffer (Millipore, Burlington, MA, USA) and centrifuged at 12,000 rpm for 30 min at 4 °C, and the supernatants were collected (n = 5/group). The hippocampal protein concentration was evaluated using a BCA protein assay kit (Thermo Fisher Scientific). The protein was adjusted to 2.0 μg/μL, isolated in 4–12% Bis-Tris Gel (Thermo Fisher Scientific), and transferred to PVDF membranes (Millipore, Bedford, MA, USA). After blocking with 1% skim milk, immunoblotting was performed with the primary antibodies summarized in Table 1. After rinsing with buffer, the immunoblotted membranes were incubated with the corresponding secondary antibodies (1:3000, Cell Signaling Technology, Danvers, MA, USA) for 60 min, and the signals were detected with an ECL kit (Cytiva, Buckinghamshire, UK). The target protein band was analyzed using an Ez-Capture MG system (Atto Corp., Tokyo, Japan), and the densitometric analysis of the bands was performed with Scion Image software (version 4.0.2, Scion Corp., Frederick, MD, USA). GAPDH was used as an internal reference to normalize the target protein expression.

Mouse lung-tissue homogenates, including metastatic nodules (n = 5/group) were lysed with radioimmunoprecipitation assay buffer (Millipore, Burlington, MA, USA) and then processed for centrifugation at 12,000 rpm for 30 min at 4 °C, and the supernatants then collected. The protein concentration was assessed and adjusted to 2 µg/µL, using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). We used 4–12% Bis-Tris Gel (Thermo Fisher Scientific, Waltham, MA, USA) for electrophoresis and then transferred the protein concentration to PVDF membranes (Millipore, Bedford, MA, USA). Immunoblotting was performed with the primary antibodies summarized in Table 1, at 4 °C overnight. The PVDF membranes were incubated with a second antibody (1:3000, CST) for 60 min and the specific protein-bands were identified on the membranes, using an enhanced chemiluminescence kit (Cytiva, Buckinghamshire, UK). The bands were captured with an Ez-Capture MG system (Atto Corp., Tokyo, Japan) and analyzed with the Scion Image software (version 4.0.2, Scion Corp., Frederick, MD, USA). GAPDH was used as a loading control for target protein normalization. The uncropped blots are shown in Supplementary Materials File S1. Densitometry readings/intensity ratio of each band are shown in Supplementary Materials File S2.