Anti notch1

Protein concentrations (n = 6) were determined in the supernatant of liver tissues by classic BCA protein assay (Beyotime). Equal protein of each sample was fractionated onto sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membrane by a Bio-Rad Western blot apparatus. The membranes were blocked with 5% fat-free milk or 5% bovine serum albumin and then probed with the following primary antibodies for 12 hours at 4°C: GAPDH (1 : 2000), Anti-Bax (1 : 2000), Anti-Bcl-2 (1 : 1000), Anti-Rheb (1 : 2000), Anti-Tuberin (1 : 1000), Anti-p-Tuberin (1 : 500), Anti-mTOR (1 : 800), Anti-p-mTOR (1 : 800), Anti-Notch1 (1 : 1000), Anti-Cyclin D (1 : 1000), Anti-p70S6K (1 : 1000), and Anti-4E-EIF (1 : 1000) (Abcam, Cambridge, UK). The membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (1 : 2000~1 : 3000, Abcam, Cambridge, UK) and visualized with an enhanced chemiluminescence (ECL) detection kit (Millipore). Bands were quantified using quantity one 4.40 software (Bio-Rad, CA, USA).

The reagent RIPA Lysis Buffer (Thermo Fisher Scientific) was used to extract the total protein of the cultivated chondrocyte or isolated cartilage tissue. RIPA was supplemented with protease inhibitor PMSF. Protein concentration was determined by a BCA kit (Thermo Fisher Scientific). Protein was mixed with protein loading buffer (Thermo Fisher Scientific) and loaded to a 12% SDS-PAGE gel for electrophoresis. Afterward, the gel was transferred to PVDF membrane (Thermo Fisher Scientific). After a blocking step with a blocking buffer, the membrane was incubated with primary monoclonal antibodies at 4 °C overnight. After washing, the membrane was incubated with secondary antibody. Finally, signal was detected by an ECL method. The primary antibodies used in this study includes anti-Aggrecan (1:1000, Abcam), anti-Collagen II (1:1000, Abcam), anti-β-actin (1:3000, Abcam), anti-ADAM8 (1:1000, Abcam), anti-p-ERK (1:1000, Abcam), anti-ERK (1:2000, Abcam), anti-p- NF-κB p65 (1:1000, Abcam), anti-NF-κB p65 (1:2000, Abcam), anti-MMP9 (1:1000, Abcam), anti-Notch1 (1:1000, Abcam), anti-Hes1 (1:2000, Abcam). All the primary antibodies were incubated for 4 h at 37 °C. The HRP-conjugated secondary antibody (1:5000, Sigma-Aldrich) was incubated for 2 h at RT.

Protein samples (50 µg) were transferred to PVDF membranes. After blocking with 5% non-fat milk for 2 h at room temperature, the membranes were incubated with primary antibodies overnight at 4°C, anti-E-Cadherin (1:2,000, Cell Signaling Technology, cat.no.14472), anti-N-cadherin (1:2,000, Cell Signaling Technology, cat.no.13116s), anti-Vimentin (1:1,000, Cell Signaling Technology, cat. no.5741s), anti-Zeb-1(1:2,000, Cell Signaling Technology, cat.no.70512s), anti-AR (1:2,000, Cell Signaling Technology, cat.no.19672s), anti-SOX17 (1:2,000, Abcam), anti-Notch1(1:2000, Abcam); anti-Notch2 (1:2,000, CST); anti-Notch3 (1:1,000, Abcam); anti-Notch4 (1:1000, Santa Cruze), and anti-GAPDH (1:1,000, CST, cat. no.5174s) was used as a loading control. The intensity of the protein bands was determined using Image-Pro plus 6.0.

The immunohistochemical staining of mouse brain sections was carried out as said previously16 (link) to study the co-expression of activated Notch1 and microglial Iba protein. After perfusion with ice-cold PBS, brains of sacrificed mouse were excised and fixed with 4% paraformaldehyde. The sections of 20 μm thick were prepared using a Leica CM3050S cryostat and after processing incubated overnight with anti-Notch1 (1:250; Abcam) and anti-Iba (1:250; Millipore) at 4 °C. Next day, after extensive washing, the sections were incubated with Alexa Fluor 488 and Alexa Fluor 594 (1:1,500; Molecular Probes, Eugene, OR) conjugated secondary antibodies. Finally, the sections were mounted with 4′-6-diamidino-2-phenylindole (DAPI; Vector Laboratories Inc., California, USA) and observed under Zeiss Apotome microscope (Zeiss, Gottingen, Germany) at the specified magnification.

All the embedded PCa and CRPC samples were cut into 5-µm-thick sections. The immunoreactivities of SOX17, Notch1, Notch2, Notch3, Notch4 were detected by an immunoperoxidase staining procedure with the primary antibodies (anti-SOX17, 1:200, Abcam, cat.no.ab192453; anti-Notch1, 1:200, Abcam, cat. no.ab8925; anti-Notch2, 1:200, Cell Signaling Technology, cat.no. D76A6; anti-Notch3, 1:200, Abcam, cat.no. ab23426; anti-Notch4, 1:200, Santa Cruz Biotechnology, cat.no. sc-377399 ). Staining scoring, according to staining intensity, was defined as 0, no staining; 1, weak staining; 2, light staining; 3, moderate staining; and 4, strong staining. Staining scores of ≤1 were defined as negative expression, while staining scores of ≥2 were defined as positive expression.