Figure 5.
Ser784 Phosphorylation Increases VCP Activity Specifically in the Nucleus
(A) HeLa cells stably expressing GFP or RNAi-resistant VCP-GFP (WT or mutants) were infected with shLuc or shVCP1 and 2 combined. Cells were analyzed 4 days later by western blot using antibodies against VCP (detecting both endogenous VCP and exogenous VCP-GFP) or actin.
(B) Cells in (A) were treated with 50 μM of etoposide for 30 min, recovered for 1 h, lysed by RIPA buffer (no SDS), and analyzed for soluble and insoluble fractions by western blot using a K48-linkage-specific polyubiquitin antibody controlled by histone H3 or GAPDH.
(C) Etoposide-treated HeLa cells, as in (B), were subjected to subcellular fractionation (Méndez and Stillman, 2000), followed by western blot analysis of the resulting cytoplasmic, nucleoplasmic, and chromatin fractions using the K48-ubiquitin antibody controlled by GAPDH, actin, and histone H3. Densitometry was performed to quantify K48-polyubiquitin levels in (B) and (C), which were subsequently normalized over the internal controls.
(D) Chromatin fractions from (C) were analyzed by western blot for Ku70.
(E) VCP knockdown and rescue HeLa cells were treated with 50 μM of etoposide for 30 min, recovered for 2 h in the presence of 20 μM of MG-132, and lysed with RIPA buffer. Soluble and insoluble fractions were analyzed by western blot for HIF1α and K48-ubiquitin, with tubulin and H3 as loading controls.
See also Figure S7.