Anti ndrg2

The cell samples or frontal lobes of brain tissues were lysed with radioimmunoprecipitation assay buffer (Thermo Scientific, 87788), and 20 μg of samples was separated with 4 to 20% SDS–polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Millipore, Shanghai, China). After blocking, the membrane was incubated overnight with the indicated primary antibody at 4°C. The following primary antibodies were used: anti-GFAP (1:10,000; Abcam, ab7260), anti-NDRG2 (1:2000; Abcam, ab174850), anti-p-Smad2 (Ser^465/467)/Smad3 (Ser^423/425) (1:1000; Cell Signaling Technology, 8828), anti-Smad2/3 (1:1000; Cell Signaling Technology, 8685), anti-Smad3 (1:1000; Cell Signaling Technology, 9528), anti–MMP-9 (1:1000; Santa Cruz Biotechnology, sc21733), anti–laminin α2 (1:200; Sigma-Aldrich, L0663), anti-occludin (1:10000; Proteintech, 66378-1), anti–ZO-1 (1:1000; Proteintech, 66452-1), anti-flag (1:5000; Sigma-Aldrich, F1804), anti-hemagglutinin (HA; 1:20000; Proteintech, 66006-2), and anti–β-actin (1:5000; Abcam, ab8226). Horseradish peroxidase–conjugated secondary antibodies were used. Chemical reactions were detected with an ECL system (Advansta, Menlo Park, CA, USA). The scanned images were analyzed with ImageJ NIH software.

The cells were incubated with an appropriate volume of immunoprecipitation lysis buffer (Pierce, Thermo Scientific) and a protease inhibitor mixture for 4 hours at 4°C. The supernatant lysates were collected and were then incubated with 2 μg of primary antibodies overnight at 4°C: anti-Flag (1:100; Proteintech, 66008-3), anti-HA (1:100; Proteintech, 51064-2), anti-NDRG2 (1:100; Abcam, ab174850), and anti-PPM1A (1:100; Cell Signaling Technology, 3549). After incubation for 4 hours at 4°C, the protein A/G magnetic beads (MedChemExpress, HY-K0202) were washed three times with the lysis buffer. The proteins were eluted in loading buffer and analyzed by immunoblotting analysis.