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. 2008 Aug 6;9(10):1743–1756. doi: 10.1111/j.1600-0854.2008.00794.x

Figure 2. Inhibition of PtdIns transfer activity and membrane retention of PITPβ following NEM treatment of HL60 cells.

Figure 2

HL60 cells were treated with 1 mmNEM for 10 min at 37°C, and cytosol and membranes were subsequently prepared. A) PtdIns transfer activity in cytosol prepared from control (closed circles) and NEM-treated cells (open circles). B) Thirty micrograms of HL60 membranes and cytosol were analysed by SDS–PAGE, followed by western blot analysis from control and NEM-treated cells. Recombinant PITPβ was run in parallel to quantify the amount of PITPβ. The values are indicated above the blot in nanograms. C and D) HL60 cytosol (180 μL) from control and NEM-treated cells was fractionated by size exclusion chromatography (Superose 12 column 10/300), and the fractions were assayed for PtdIns and PtdCho transfer activity (inset) (C) and PITPβ distribution by western blot analysis (D). The column was calibrated using a kit containing proteins of molecular weight 200, 67, 43, 25, and 13.7 kDa, and their elution profile is indicated in (D). PITPβ in the cytosol prepared from NEM-treated cells elutes as a 67-kDa protein.