Fig 3.

Proteasome inhibition increases the abundance of ubiquitinated Bax in MAM (A) but not in mitochondria (B) of HCMV-infected cells. HFFs were uninfected or infected with HCMV wt (MOI of 3). Cells were treated with the proteasome inhibitor MG132 (2.5 μM) from 48 to 72 hpi. Uninfected (4 roller bottles) and HCMV-infected (4 roller bottles) HFFs were harvested and fractionated as described previously (22, 66). MAM proteins (200 μg) (A) or mitochondrial proteins (200 μg) (B) were immunoprecipitated (IP) with ubiquitin affinity resin (Pierce ubiquitin enrichment kit; Thermo Scientific). Immunoprecipitated proteins were resolved by SDS-PAGE and examined by Western blot (B) analyses for the presence of Bax (left), vMIA (middle), or total ubiquitinated proteins (right). In panel B, a longer exposure (1 min) of the mitochondrial proteins from untreated, uninfected, and HCMV-infected cells is shown below the original exposure (5 s), corresponding to the exposure time of mitochondrial proteins from MG132-treated cells (5 s).