Figure 1.

(A) SREBP-2 is actively imported into the nuclei of living cells. Purified recombinant FLAG-SREBP-2 (0.5 mg/ml) was injected into the cytoplasm of HeLa cells with (b) or without (a and c) 2 mg/ml WGA. After incubation for 30 min at 37°C (a and b) or on ice (c), cells were fixed, and the localization of FLAG-SREBP-2 was detected by indirect immunofluorescence with an anti-FLAG M2 antibody. (B) Nuclear import of SREBP-2 requires soluble factors. Digitonin-permeabilized HeLa cells were incubated with 10 μl of a reaction mixture containing 4 pmol of FLAG-SREBP-2 with ATP regeneration system only (a), 4 mg/ml cytosol and an ATP regeneration system (b and d), or 4 mg/ml cytosol and 18 U of hexokinase (c). WGA pretreatment was performed by incubating the cells with 0.5 mg/ml WGA for 5 min on ice before incubating with reaction mixture (d). Import reactions were performed for 20 min at 30°C, and FLAG-SREBP-2 was detected by indirect immunofluorescence.