X ray processor

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).

For inhibitor studies on the degradation of CPE-QQ and CPE-WT, equivalent amounts of N2a cells were stably transfected with human CPE-WT, CPE-QQ or EV (pcDNA3.1, negative control) cDNAs. Cells were then treated with a proteasomal inhibitor, MG132 (5 μm, Sigma, St Louis, MO, USA) or a lysosomal inhibitor, E64d (20 μm, Sigma), for 24 h and then collected for western blot analysis. The cells were extracted with M-PER lysis buffer, quantified by the Bradford Assay (Thermo Scientific) and equal protein amounts were analyzed by western blot with a mouse anti-CPE monoclonal antibody (1:10 000, R&D Systems, Minneapolis, MN, USA) and then with a mouse anti-β-actin monoclonal (1:2000, Cell Signaling, Danvers, MA, USA) antibody. The bands were visualized with luminal:peroxide ECL substrate (Thermo Scientific). A Kodak X-Ray processor was used for fixing and developing the signals on the X-ray films. The bands were quantified by using Fuji (Image J) program (background subtracted). In similar experiments, cell extracts were analyzed by western blot for levels of CHOP, an ER stress-induced transcription factor.