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. 2010 Dec 1;21(23):4141–4150. doi: 10.1091/mbc.E10-05-0424

Figure 1.

Figure 1.

Analysis of an oxysterol-activated PI4K in the Golgi and endosomes. (A) Homogenates of CHO cells, cultured in the absence (−) or presence (+) of 25-hydroxycholesterol (25OH; 2.5 μg/ml) for 1 h, were fractionated on discontinuous sucrose gradients. Equivalent volumes of fractions I–V were resolved by SDS-PAGE and immunoblotted for the indicated proteins as described in Materials and Methods. (B) Sucrose gradient fractions of untreated CHO cell homogenates were assayed for PI4K activity and expressed as a percentage of total. Results are the mean and SEM of three experiments. (C) Homogenates (H) of CHO cells treated with 25-hydroxycholesterol (2.5 μg/ml) or control solvent for 1 h were separated on discontinuous sucrose gradients, and individual fractions were assayed for PI4K activity and expressed relative to controls. Results are the mean and SEM of three experiments. (D) Fraction II was isolated from cells treated with 25-hydroxycholesterol for the indicated times and assayed for PI4K activity. Results are expressed relative to time-matched untreated controls (mean and SEM of 6 experiments).