Horseradish peroxidase labeled secondary antibody sc 2004 sc 2005

Whole-cell lysates were prepared from CLL cells using RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA). After quantification, an equal amount of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins in the gel were transferred to nitrocellulose membranes. Next, the membranes were incubated overnight at 4 °C with primary antibodies against FOXO1, p53, and CDK6 (#2880, #2524, and #3136, respectively, Cell Signaling Technology, Danvers, MA, USA) (19 (link)); Bax, TCF4, MYD88, and TLR4 (ab7977, ab185736, ab2064, and ab13556, respectively, Abcam, Cambridge, UK), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, sc-32233, Santa Cruz Biotechnology, Dallas, Texas, USA). The membranes were then probed with a horseradish peroxidase-labeled secondary antibody (sc-2004/sc-2005, Santa Cruz Biotechnology) for 1 h at room temperature. Finally, protein signals were detected through chemiluminescence using ECL Western Blotting Detection Reagents (GE Healthcare, Pittsburgh, PA, USA) and visualized through the G:BOX Chemi Gel Documentation System (Syngene, Frederick, MD, USA).