Figure 3.

Paip2 abundance in response to HCMV infection, serum stimulation, or UL38 induction is regulated by mTORC1. (A) Asynchronous NHDFs were either mock-infected or HCMV-infected in the presence of DMSO, the mTORC1-selective inhibitor rapamycin (100 nM), or the active site mTOR inhibitor PP242 (2.5 μM). At 48 hpi, total protein was collected, fractionated by SDS-PAGE, and analyzed by immunoblotting with the indicated antisera. (B, lanes 1–4) NHDFs that express UL38 upon induction with dox cells were growth-arrested by serum deprivation; treated for 1 h with DMSO, rapamycin (100 nM), or PP242 (2.5 μM); and subsequently untreated or serum-stimulated with 20% FBS for 20 min in the presence or absence of drug. (Lanes 5–7) Alternatively, cells were dox-treated for 72 h. Subsequently, cultures were metabolically pulse-labeled for 1 h with [³⁵S]Met-Cys, and total protein was collected. A sample of the lysate was immunoprecipitated using anti-Paip2 antisera. Lysates (input panel) and isolated immune complexes (Paip2 panel) were fractionated by SDS-PAGE, and radiolabeled polypeptides were directly visualized by exposing the fixed, dried gel to X-ray film. The migration of molecular weight standards (in kilodaltons) is shown to the left of the top panel.