Anti p mdm2

The primary antibodies used in this study included anti-Akt, anti-p-Akt, anti-CCND1, anti-E2F1, anti-p-MDM2, and anti-P27 (Cell Signaling Technology, USA) and anti-TRIB3, anti-E-cadherin, anti-MMP9, anti-H3 and anti-GAPDH (Abcam, USA), and anti-MDM2 (Proteintech, USA). The goat anti-rabbit or anti-mouse secondary antibodies were purchased from Kangwei Ltd. Beijing, China.
To determine the protein levels in cells, total protein was extracted using RIPA with a Protease Inhibitor Cocktail. Then, 30 µg of protein was subjected to 10% SDS–PAGE gel electrophoresis and transferred to a polyvinylidene fluoride membrane. After the membrane was blocked with 5% skim milk for 1 h, blots were incubated with primary antibodies. Finally, the blots were incubated with secondary antibodies after being washed with TBST.

For all Western blotting analyses, HAECs were collected and lysed in a radioimmunoprecipitation assay (RIPA) buffer (Pierce, Rockford, IL, USA) containing a protease inhibitor cocktail. The protein concentrations were determined using a BCA assay (Pierce). Samples were separated by SDS-PAGE (4–20% gels; Invitrogen, Carlsbad, CA, USA) and transferred to a PVDF membrane. Primary antibodies, including anti-FBXO31 (A302-047A, Bethyl Laboratories, Montgomery, TX, USA, 1:1000), anti-p-MDM2 (#3521, Cell Signaling Technology, 1:1000), anti-p53 (#2524, Cell Signaling Technology, 1:1000), anti–p21 (GTX62525, GeneTex, Alton Pkwy Irvine, CA, USA, 1:1000), and anti-β-actin (A5441, Sigma-Aldrich, 1:5000), were used. All signals were detected using an ECL reagent (Millipore, Bedford, MA, USA). Statistical results were obtained by scanning the reactive bands and measuring the density of optical cells using video densitometry (G-box Image System, Syngene, Frederick, MD, USA) [23 (link)].

Cells were harvested and rinsed with PBS, and lyzed in denaturing lysis buffer (Applygen Technologies Inc. China) for 30 min on ice, centrifuged 12,000 g for 20 min at 4 °C. Protein concentrations were determined by BCA assay. Equal quantities (30 μg of protein) of cell extract were resolved by 10% SDS-PAGE, the resolved protein were electrophoretically transferred to PVDF membrane, and blocked with 5% fat-free dry milk in TBST for 1 h at room temperature. The membrane was immunoblotted with anti-γ-H2AX, anti-p21, anti-p-ATM, anti-ATM, anti-p-Mdm2, anti-Mdm2, anti-p-p53, anti-AKT, anti-β-actin (Cell Signaling Technology, USA), anti-p53, anti-Bax and anti-Bcl-2 (Santa Cruz, USA) antibodies in 5% milk TBST, at 4 °C overnight. The membranes were washed 3 times, incubated with HRP-conjugated secondary antibodies for 1 h at room temperature, and washed extensively before detection. The membranes were subsequently developed using ECL (FujiFilm, Japan) reagent (Applygen Technologies Inc. China) and exposed to film according to the manufacturer’s protocol.