Fig. 5: Cyclin B1-NT is efficiently proteolyzed in a ubiquitin-independent pathway.
From: The 20S as a stand-alone proteasome in cells can degrade the ubiquitin tag
a Cloning strategy for expression of HA-Cyclin B1-NT chimera fused to the NS3-protease in a mammalian expression vector (left). The resulting fusion protein of cyclin B1 N-terminus (residues 1–88 aa) with the NS3-protease (right). b HEK293T cells expressing HA-Cyclin B1-NT were treated with cycloheximide (CHX; 50 μg/mL) and/or bortezomib (1 μM) and/or chloroquine (CQN; 50 μM) for 2 h followed by IB for the indicated proteins. c HEK293T cells expressing HA-Cyclin B1-NT were treated with cycloheximide (50 μg/mL) and/or bortezomib (1 μM)/E64d (10 µM)/z-VAD-FMK (50 µM)/CHR-2797 (10 µM)/chloroquine (50 μM) for 2 h followed by IB for the indicated proteins. d HEK293 cells expressing HA-tagged Cyclin B1-NT were treated with 50 µg/mL cycloheximide for 1 h, with or without E1 inhibitor (PYR-41) at 25 µM and IB for the indicated proteins. e HA-Cyclin B1-NT expressing HEK293T cells were treated with/without bortezomib (1 μM) and/or PYR-41 (10 μM) for indicated time periods followed by IB as indicated. f HA-Cyclin B1-NT expressing HEK293T cells were treated with bortezomib (1 μM) and/or PYR-41 (10 μM) for 6 h followed by immunoprecipitation of HA-Cyclin B1-NT using anti-HA antibody. IB was performed using anti-ubiquitin antibody for immunoprecipitated fraction and anti-Cyclin B1-NT for 5% Input. g HEK293T cells expressing either HA-tagged WT- or K0-Cyclin B1-NT were treated with a pulse of bortezomib at 0.5 µM concentration for 6 h followed by cycloheximide chase. Various proteins in cell lysates were detected by IB. h Wild type (WT) or K0-Cyclin B1-NT were trans-expressed in HEK293T cells followed by 6 h pulse treatment of bortezomib (0.5 μM). HA-Cyclin B1-NT was immunoprecipitated using anti-HA antibody. Elution of immunoprecipitated fraction was IB using anti-ubiquitin antibody, 5% of Input was IB using anti-Cyclin B1-NT. i K0-Cyclin B1-NT expressing HEK293T cells were pulse treated with bortezomib (0.5 μM) for 6 h. Then bortezomib was removed and cells were grown with/without treatment of cycloheximide (50 μg/mL) and/or PYR-41 (10 μM) for indicated time periods followed by IB as indicated. j K0-Cyclin B1-NT expressing HEK293T cells were pulse treated with bortezomib (0.5 μM). Then bortezomib was removed and cells were grown with/without treatment of cycloheximide (50 μg/mL) and/or bortezomib (1 μM)/chloroquine (50 μM) for 2 h followed by IB as indicated. Source data are provided as a Source data file.
