Protein Protocols Protein Protocols

648 Corsini
Table 1
Mevalonate, Prenols, and Synthetic Analogs Vary in Their Ability
to Prevent the Inhibitory Effect of Simvastatin on DNA Synthesisin
Cultured Cellsas Measured by Incorporation of [ 3 H]-Thymidine
Cell type HSF 3T3
Treatment Conditions: (Reported as % control)
Simvastatin (S) 40 µM 30 30
S + mevalonate 100 µM 100 90
S+ all-trans-GGOH 5 µM 85 96
S+ 2-cis-GGOH 5 µM 29 30
S+ all-trans-FOH 10 µM 37 33
S+ geraniol 5 mM 29 30
S+ 6,7,10,11,14,15-hexahydro-GGOH 5 µM 34 46
S+ tetrahydro FOH 5 µM
Experimental conditions: see Subheading 3.2.
N.A., not assayed.
N.A. 36
The mean value of control (100%) without inhibitor was 104 × 103 ± 3 × 103 dpm/plate
and 187 × 103 ± 10 × 103 dpm/plate for HSF and 3T3 cells, respectively.
Fig. 4. The ability of simvastatin-treated HSF cells to synthesize DNA was used as a measure
of their ability to traverse the cell cycle. HSF cells were seeded at a density of 3 × 10 5 /35-mm
dish in medium supplemented with 0.4% FCS, and the cultures were incubated for 72 h. Quiescent
cells were incubated for 24 h in fresh medium containing 10% FCS, 40 µM simvastatin, and
6. Add 1 mL chloroform:methanol (2:1), mix, sonicate, and incubate for 30 min at 37°C.
Centrifuge for 5 min (13,000 g, 4°C), and collect the lipid extracts.
7. Remove the residual organic solvent by evaporation, and solubilize the delipidated proteins
overnight at room temperature in 100 µL 3% SDS, 62.5 mM Tris-HCl, pH 6.8.
8. Determine the protein content in 10 µL of sample, according to Lowry (27).
9. Transfer 10 µL of the sample to a liquid scintillation vial, add 5 mL Aquamix, and measure
the incorporated radioactivity.
10. Add 80 µL of sample application buffer to the remainder, and analyze an aliquot (15–80 µg
of protein) by one-dimensional SDS-PAGE, according to Laemmli (28), using a 12% gel.