FIGURE 2.

Activation of typical and atypical neddylation by multiple stresses in cardiomyocytes. A, NRVMs were treated with the proteasome inhibitor bortezomib (BZM) for the indicated times. HMW, high molecular weight; conj, conjugate. B, NRVMs were treated with the proteasome inhibitors BZM (100 nm), MG132 (5 μm), and MG262 (1 μm) for 6 h. The NAE inhibitor MLN4924 (MLN) was added 1 h after the administration of proteasome inhibitors. Veh, vehicle. C, NRMMs were treated with vehicle or BZM (100 nm) in the absence or presence of MLN4924 (1 μm) for 6 h. Polyubiquitinated proteins were purified from cell extracts using TUBEs under denaturing or non-denaturing conditions and immunoblotted with the indicated antibodies. D, NRMMs were subjected to sI for 12 h. E, NRMMs were infected with Ad-SF-NEDD8 for 24 h and treated with H₂O₂ for another 8 h. A–E, Shown are representative Western blots and the densitometric analysis from three independent experiments. The asterisks and # denote high and low molecular weight NEDD8 conjugates respectively.