Figure 9.
IGF-1 induces SREBP processing and ER-to-Golgi transport of GFP-SCAP, which is inhibited by LY294002 and 25-hydroxycholesterol. (A) 13A/PS cells were cultured in medium E for 24 h before the incubation for 16 h in medium E containing various concentrations of IGF-1 in the absence or presence of LY294002 (20 μM). Medium was assayed for PLAP secretion by luminometry, and cell protein was determined. Values are means + SEM (from n = 3 replicate cultures, representative of 3 separate experiments). (B) 13A/PS cells were cultured in medium E for 24 h before the incubation for 16 h in medium E in the absence or presence of IGF-1 (50 nM) and were coincubated in the absence or presence of LY294002 (20 μM) or 25HC (1 μg/ml). Total cell lysates (containing 50 μg protein) were subjected to 7.5% SDS-PAGE and immunoblot analysis with anti-SREBP-2 IgG-7D4 as described in Materials and Methods. P and N denote the precursor and cleaved nuclear forms of SREBP-2, respectively. Relative intensity of the N band relative to the total (N+P bands) was quantified by densitometry and presented relative to the vehicle-treated control (lane 1). (C) Stably transfected CHO/pGFP-SCAP cells were set up on glass coverslips as described in Materials and Methods. On day 1, cells were cultured in medium E for 24 h. On day 2, cells were switched to fresh medium E and treated for 16 h in the absence or presence of IGF-1 (50 nM) and were coincubated with LY294002 (20 μM) or 25HC (1 μg/ml). Representative fields are shown for each condition from repeated (n = 3) experiments. Parental cells (SRD-13A) lacking GFP-SCAP showed no fluorescence under these conditions (unpublished data).