Figure 3.

Visualizing InsP₆, InsP₇ and InsP₈ from mammalian cells and organs. (a) Cells from six different human lines were collected and washed in PBS. A small aliquot was taken for determination of protein concentration, while the rest was PA extracted and subjected to TiO₂ bead enrichment. Extracts relative to equivalent amounts of protein for each cell line (approx. 35 mg) were loaded on two parallel gels subsequently stained by DAPI (top) and toluidine blue (bottom). (b) Densitometry of toluidine blue-stained gel from three independent experiments was used to calculate ratios of InsP₇ and InsP₈ over their precursor InsP₆. (c) Mouse brain and liver (approximately 0.5 g) were homogenized and extracted with PA. After TiO₂ purification the inositol phosphates were resolved by PAGE and stained with DAPI. (d) Two 14 cm dishes of 80% confluent HCT116 cells were pre-treated in glucose-free medium for 30 min before addition of 5 µM oligomycin for 3 h. The TiO₂ extracts were then resolved by PAGE and stained with toluidine blue. The gels presented are representative of experiments performed three times.