Hrp conjugated secondary ab

Subchondral bone tissues or RAW 264.7 cells were lysed in RIPA buffer (Solarbio) supplemented with PMSF, a phosphatase inhibitor, and protease inhibitor (Sigma-Aldrich). The lysates were centrifuged at 4°C, 12,000g for 20 minutes. Total protein concentration was measured by bicinchoninic acid Protein Assay Kit (Thermo Scientific). The aliquot of proteins was subjected to SDS-PAGE and transferred electrophoretically to polyvinylidene fluoride membranes (Millipore). After blocking with 5% nonfat milk, the membranes were incubated with primary Ab, including p-Akt1 (1:1000; Cell Signaling Technology, 9018), Akt1 (1:1000; Cell Signaling Technology, 2938), p-Akt2 (1:1000, Cell Signaling Technology, 8599), Akt2 (1:1000, Cell Signaling Technology, 3063), CTSK (1:1000; Abcam, ab19027), RANKL (1:1000; Abcam, ab62516), OPG (1:1000; ABclonal, A13250), and β-actin (1:10000; Proteintech, 66009-1-lg). After washing, the blots were probed with HRP-conjugated secondary Ab (1:10,000; Cell Signaling Technology) and subjected to enhanced chemiluminescence detection (Thermo Fisher Scientific). The intensity of bands was quantified by ImageJ and normalized to the density of the internal control.

After treating with different concentrations of juglone derivatives for 48 h, total protein of glioma cell lines of U87 and U251 were obtained from RIPA lysis buffer with 1% PMSF (Beyotime, China). The protein concentration was determined by BCA assay (Beyotime, China), and samples were separated on 10% SDS-PAGE, and then transferred onto NC membranes (0.45 μm, Millipore, United States). The membranes were incubated with primary Abs against cleaved-PARP (1:1000, Cell Signaling Technology, China) and β-actin (1:10000, Cell Signaling Technology, China) overnight at 4°C, followed by HRP conjugated secondary Ab (1:3000, Cell Signaling Technology, China). The protein bands were visualized using enhanced chemiluminescence (ECL, Millipore, United States) and a detection system (ChemiDoc Touch, Bio-Rad)