Phospho akt and akt

Apoptotic markers such as Bcl-xl, Bax, Bcl-2, PI3K, P38 and JNK were examined by western blot. Shortly, 1 × 10⁶ HeLa cells were uncovered with [Cu(PMPP-SAL)(EtOH)] or DMSO for 24-h incubation. Then, the proteins were extorted from cells using RIPA buffer. The protein concentration was measured using Pierce™ Detergent Compatible Bradford Assay Kit (USA). Then, the proteins were positioned to 15% SDSP-PAGE and the gel was reassigned to PVDF membranes. The membrane was blocked with 5% bovine serum albumin for 3 h. The membranes were combined with a suitable primary antibody and kept for 24-h incubation at 4 °C. PARP, Cyt c, Bcl-2, Bax and β-actin antibodies were from Cell Signaling Technology Co. JNK, phospho-JNK, IκBα, p38, phospho-p38, phospho-AKT and AKT antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Then membranes incubated with suitable conjugated horseradish peroxidase secondary antibodies for 1 h. Anti-mouse IgG-HRP and anti-rabbit IgG-HRP were purchased from Santa Cruz Biotechnology Co. Protein bands were detected using the ECL assay kit (Beyotime, Jiangsu, China) and exposed using a Kodak medical X-ray processor (Kodak, USA).

Protein extraction from pulverized tissues was performed using the following lysis buffer: 10 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1 mM EDTA; 1 mM EGTA; 0.1% SDS; 1% Nonidet P-40; 1 mM PMSF, 10 μg/mL leupeptin, 100 μg/mL aprotinin, 10 mM benzamidine, 1 mM NaF, 1 mM sodium orthovanadate, and 1 mM DTT. The cell lysates were swirled for 30 minutes at 5 °C and centrifuged at 13500 rpm for 15 minutes (Eppendorf Centrifuge 5417 R). The supernatant was subsequently analyzed for total protein content using the Bradford quantification method (Bio-Rad, Hercules, CA). Fifty micrograms of protein was loaded into 12% acrylamide gels and separated by SDS-PAGE. Proteins were subsequently transferred onto a nitrocellulose membrane, blocked with 1% BSA and incubated with antibodies against VEGF (Abcam), β-actin (Millipore), phospho-ERK1/2 and ERK2 (Santa Cruz Biotechnology), phospho-Akt and Akt (Cell Signaling). Anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibodies and an ECL kit were used to visualize immunoblots. Quantification by densitometry was performed using ImageJ software49 (link).

Western blot analysis was performed as previously described [31 (link)]. Antibodies against phospho-Akt and Akt (Cell Signaling Technology, Danvers, MA), phospho-ERK and ERK (Cell Signaling Technology) were used, and signals were visualized by chemiluminescent detection according to the manufacturer’s protocol (Amersham Pharmacia Biotech, London, UK).