Chemiluminescent enhanced chemiluminescence assay kit

Total cell lysates were obtained with an extraction buffer as previously described. Protein concentrations were determined using a protein assay kit (Bio-Rad, Hercules, CA, USA). For Western blot analysis, the cell lysates were separated by 12% SDS-PAGE, transferred into a polyvinylidene fluoride membrane (GE Healthcare), blocked with 5% skim milk, and incubated with the primary antibodies (1:1000 dilution). Antibodies against caspase-3, -8, -9, Bax, Bcl-2, Bcl-xL, HIAP-1, HIAP-2, p53, p21, E2F1 and p73 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). After incubation with the horseradish peroxidase-conjugated secondary antibody at room temperature, immunoreactive proteins were detected using a chemiluminescent enhanced chemiluminescence assay kit (GE Healthcare) according to the manufacturer’s instructions. Bands in the blot were visualized using a LAS3000 luminescent image analyzer (Fujifilm Life Science, Tokyo, Japan) [15 (link)].

Protein lysates were added into the tissues after rinsing with pre-cooled PBS for 3 times, lysed at 4 °C and centrifuged (10,000 r/min) for 15 min. Supernatant proteins were then extracted and mixed with sodium dodecyl-sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) loading buffer. Primary antibodies were put into them after SDS-PAGE gel electrophoresis and transfer to a membrane, and the proteins were placed overnight at 4 °C. Then horseradish peroxidase-conjugated secondary antibodies were incubated with the proteins at room temperature. In the end, immunoreactive proteins were tested with a chemiluminescent enhanced chemiluminescence assay kit (GE Healthcare, Uppsala, Sweden) and observed with a LAS3000 luminescent image analyzer (Fujifilm, Tokyo, Japan) with β-actin as internal reference [39 (link)].

Protein lysates were added into the gastric tissues after rinsing with pre-cooled PBS 3 times, lysed at 4 °C and centrifuged (10,000 r/min) for 15 min. Supernatant proteins were then extracted and mixed with SDS-PAGE (sodium dodecyl-sulfate-polyacrylamide gelelectrophoresis) loading buffer. Primary antibodies were put into them after SDS-PAGE gel electrophoresis and transfer to a membrane, and the proteins were placed overnight at 4 °C. Then horseradish peroxidase-conjugated secondary antibodies were incubated with the proteins at room temperature. Finally, immunoreactive proteins were tested with a chemiluminescent enhanced chemiluminescence assay kit (GE Healthcare, Uppsala, Sweden) and observed with a LAS3000 luminescent image analyzer (Fujifilm, Tokyo, Japan) with β-actin as internal reference [8 (link)].