Biovision ecl western blotting substrate kit

After 6 h, 24 h and 48 h treatment of 4T1 cells with memantine, cells were washed with PBS and scraped into the RIPA lysis buffer containing 1mM PMSF followed by sonication for 15 seconds. Samples were centrifuged for 15 minutes at 14000 rpm at 4 °C and the supernatant was collected. Proteins were quantifi ed by using the BCA Assay Kit (Thermo Pierce, Rockford, IL, USA). Protein lysates (20 μg) were heated for 5 minutes at 95 °C in LDS non-reducing sample buffer (Pierce, Rockford, IL, USA) and then loaded into the 10 % Tris-glycin gels. The gels were transferred to the PVDF membrane (Merck Millipore, Darmtadt, Germany) at 250 mAmp for 90 minutes. Membranes were blocked with 5 % non-fat milk powder in TBS-T for 1 hour at room temperature and incubated overnight at 4 °C with the primary antibodies for Bax, Bcl-2, Casp-3, Casp-9, E-Cad, Vimentin, B-Cat, GSK3B, p-ERK, ERK, p-GS, GS and Gapdh at 1:1000 dilution (Thermo Pierce, Rockford, IL, USA). Blots were washed with TBS-T subsequently. Protein bands were detected by using the secondary antibody (Thermo Pierce, Rockford, IL, USA) and the blots were visualized by BioVision ECL Western Blotting Substrate Kit (Biovision, California, USA). Chemiluminescent signals of immunob-lots were detected by using the Gel Logic 2200 Pro (Carestream Health, Rochester, NY, USA).