Anti stat3

Sorted CD133+ cells were treated with McCoy's 5A containing 0 μM, 0.625 μM, or 1.25 μM stattic for 24 h. Thereafter, protein lysates were collected and a protein assay (BioRad) was performed. The concentration of total protein in cells was quantified using the bicinchoninic acid (BCA) protein assay reagent following the manufacturer's instructions. Cells were lysed with MPER (Pierce) supplemented with protease and phosphatase inhibitors for 30 min on ice, and lysates were clarified by centrifugation for 5 min at 12,000 /g. Twenty-five to fifty milligrams of lysates were loaded onto 5% SDS-PAGE gels and were transferred onto PVDF membranes. The membranes were blocked with 5% skim milk for 1 h and were incubated overnight at 4°C with anti-Bcl-2 (ImmunoWay, USA), anti-Bcl-xl (ImmunoWay, USA), anti-P-STAT3 (ImmunoWay, USA), anti- STAT3 (ImmunoWay, USA) antibody, anti-β-actin (ImmunoWay, USA) antibody (1/1000 dilution) was used as the internal loading control. The membranes were washed three times and then were incubated with a secondary antibody (1/1000 dilution) at room temperature for 1 h. Finally, the membranes were washed three times and were exposed in the dark room; ImageJ software was used for image analysis.