Cycloheximide chase assay.

HEK 293T cells were split in 60-mm dishes. The next day, they were transfected with 1 μg of pCDNA-topo-UNC5B and 2 μg of pCDNA3.1-PyST-HA-FLAG constructs. After approximately 40 h posttransfection, cycloheximide was added at a concentration of 25 μg/ml for different time periods (3, 6, 9, and 12 h). Cell lysates were obtained using cell NP-40 lysis buffer containing protease and phosphatase inhibitors and subjected to Western blotting as described above. Data obtained after densitometry analysis using Image Studio Lite ver 5.2 (LI-COR Biosciences) were used for making graphs for comparing protein amounts from different samples. Statistical analysis was performed using GraphPad Prism software.