Mouse polyclonal anti gapdh

Whole-cell lysates were obtained using the ProteoJET™ Mammalian Cell Lysis Reagent (Thermo Scientific, Rockford, IL, USA). Proteins (50 μg) were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. The membrane was blocked with 5% non-fat dried milk in TBST (50 mM Tris [pH 7.5], 200 mM NaCl, 0.05% Tween 20) at room temperature for 1 h. The membrane was then incubated with primary antibody in the same concentration of milk in TBST for 1 h at room temperature, washed three times with TBST for 15 min, and then incubated with the HRP-conjugated secondary antibody at room temperature for 1 h. The membrane was again washed three times with TBST. Antigen-antibody complexes were detected using the SuperSignal detection reagents (Thermo Scientific, Rockford, IL, USA). The following antibodies were used: rabbit monoclonal anti-p53, rabbit polyclonal anti-Phospho-p53, mouse polyclonal anti-GAPDH, mouse polyclonal anti-ACTIN (Cell Signaling Inc. Danvers, MA, USA); secondary antibodies (goat-anti-rabbit and goat-anti-mouse) conjugated to horseradish peroxidase (Santa Cruz. CA, USA).

Shikonin (assay 99.8%) was obtained from Shanghai yuanye Bio-Technology Co., Ltd. (Shanghai, China). DSPE-PEG2000-RGD (purity > 97%) was synthesized by Xi'an ruixi Biological Technology Co., Ltd. (Xi’an, China). DSPE-PEG2000 was purchased from ToYong Biotch Co., Ltd. (Shanghai, China). Egg phosphatidylcholine (EPC, assay 98%) was obtained from LIPOID GmbH (Ludwigshafen, Germany). Cholesterol (Chol), sephadex G-50, coumarin-6 (Cou6), and Hoechst 33258 were all purchased from Sigma-Aldrich (Shanghai, China). Annexin V–FITC/PI apoptosis detection kit was purchased from Beijing ComWin Biotech Co., Ltd. (Beijing, China). 3-(4,5-Dimethylth-iazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Gibco Brl (Shanghai, China). Rabbit polyclonal anti-human-MMP-2 and rabbit polyclonal anti-human-MMP-9 were obtained from Proteintech Group, Inc. (Chicago, IL, USA). Rabbit polyclonal anti-human-NF-κB p65, mouse polyclonal anti-human-Bcl-2, mouse polyclonal anti-human-Bax, mouse polyclonal anti-GAPDH, rabbit anti-mouse immunoglobulin G (IgG)and goat anti-rabbit IgG were all purchased from Cell Signaling Technology, Inc. (Boston, MA, USA).