Figure 8.

Dominant-negative Akt also disrupts ER-to-Golgi transport of GFP-SCAP in CHO cells and inhibits SREBP-2 processing. (A–F) Stably transfected CHO/pGFP-SCAP cells were set up on coverslips as described in Materials and Methods. On day 1, cells were transfected with dominant-negative Akt (DN-Akt; A–C) or wild type-Akt (WT-Akt; D–F). On day 2, cells were cultured in medium B for 4 h in the presence of compactin (5 μM). Immunofluorescence staining was performed as described in Materials and Methods. Representative fields are shown for each condition from repeated (n = 5) experiments. Parental cells (SRD-13A) lacking GFP-SCAP showed no fluorescence under these conditions (unpublished data). (G) SCAP-null SRD-13A cells were transiently transfected with pCMV-PLAP-BP2 (0.25 μg), pTK-SCAP (0.05 μg), DN-Akt (0.5 μg), or WT-Akt (0.5 μg) in medium A for 24 h. Cells were incubated in medium B for 16 h in the presence or absence of compactin (CPN, 5 μM). Medium was assayed for PLAP secretion by luminometry, and cell protein was determined. Values are means + SEM and are representative of three separate experiments.