Pulks757

Antibodies against LKB1, pLKB1^S428, AMPK, pAMPK^Thr172, p70S6K, pp70S6K^Thr389, S6 Ribo, pS6 Ribo^S235/236, pS6 Ribo^S240/244, ULK1, pULK1^S555, pULK^S757, LC-3B, cyclin A, cyclin D1, and PARP were purchased from Cell Signaling. Antibodies for p27, cyclin B1, and CDC25C were from Santa Cruz Biotechnology, and β-actin from Sigma-Aldrich. Secondary antibodies were purchased from GE Healthcare. Cells were treated with indicated concentrations of hMGL-4.0 for the specified time. After incubation, cells were washed with ice-cold PBS and lysed in radioimmunoprecipitation assay buffer. Proteins were separated by using 4 to 15% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. After blocking in 5% BSA for 1 h, the membranes were probed with specific primary antibodies (listed above) overnight at 4 °C. Following secondary antibody incubation, the membranes were visualized using a commercially available chemiluminescent detection kit (Pierce Biotechnology).