Fig. 7: Hypoxia-induced 20S proteasome levels and accelerated Cyclin B1-NT degradation.
From: The 20S as a stand-alone proteasome in cells can degrade the ubiquitin tag
a HeLa cells were grown under normoxia (21% O2) or hypoxia (1% O2) for the indicated time periods. Cell lysates were resolved by 4% native gel followed by proteasome in-gel activity assay or transferred for IB of native complexes using the indicated proteasome antibodies (the 19S RP subunit PSMD1, PSMD2 and 20S CP subunits PSMA1). b The calculated relative distribution of 30S, 26S, and 20S proteasome species in normoxic or hypoxic cells. Data represent the mean percentage of proteasome content (±SD error bar) calculated from PSMA1-native IB of three experiments at each time period. c The lysates of cells under hypoxia (as in a) were resolved by SDS-PAGE followed by IB to evaluate proteasome subunits (HIF1-α is a marker for hypoxia, GAPDH is a loading control). d The cartoon illustrates proteasome disassembly under hypoxia into 20S and 19S sub-complexes. e HEK293T cells were either grown under normoxia or hypoxia (1% O2) for 24 h. Cell lysates were resolved by 4% native gel for proteasome in-gel activity assay or transferred for immunoblot (IB) using anti-PSMA1 antibody. f HEK293T cells grown under normoxia or hypoxia for 24 h were transfected with WT- and K0-Cyclin B1-NT plasmid constructs, then treated with 0.5 µM bortezomib followed by cycloheximide chase. Cyclin B1-NT levels were detected by IB. g Line graph represents WT- and K0-Cyclin B1-NT protein levels under conditions as in (f) measured from anti-cyclin B1 antibody IB, under both normoxia and hypoxia at each time point of cycloheximide treatment. Error bars (±SD) were calculated from the value of three independent experiments. Source data are provided as a Source data file.
