Protein Protocols Protein Protocols

644 Corsini
8. Simvastatin in its lactone form (gift from Merck, Sharp and Dohme; Rahway, NJ) is dissolved
in 0.1 M NaOH (60°C, 3 h), to give the active form. Adjust the pH to 7.4 and the
concentration to 50 mM, then sterilize by filtration.
9. Swiss 3T3-albino mouse cell line (3T3) were from American Type Culture Collection
(ATCC), Rockville, MD.
10. Human skin fibroblasts (HSF) are grown from explants of skin biopsies obtained from healthy
individuals. The cells are used between the fifth and fifteenth passages.
2.3. One-Dimensional and Two-Dimensional Gel Electrophoresis
1. SDS, TEMED, ammonium persulfate, mol wt protein standards, glycine, Bradford protein
assay kit, heavyweight filter paper, and Bio Gel P6-DG (Bio-Rad, Hercules, CA).
2. Duracryl preblended acrylamide solution (30% T, 0.65% C, used for both one-dimensional
and 2DE gels) and all other 2DE gel reagents, were obtained from ESA, Chelmsford, MA.
3. Ethylmaleimide, N-[ethyl-1,2- 3 H] ([ 3 H]-NEM), 57 Ci/mmol (NEN).
4. N-ethylmaleimide (Aldrich).
5. Soybean trypsin inhibitor and SDS-7 mol wt standards used with 2DE gels (Sigma).
6. Amplify and Hyperfilm (Amersham).
7. Reacti-Vials (Pierce, Rockford, IL).
8. DNase1 and RNaseA (Worthington Biochem, Freehold, NJ).
3. Methods
3.1. Synthesis and Labeling of Prenols and Analogs
3.1.1. Synthesis of Racemic 6,7,10,11,14,15 Hexahydrogeranylgeraniol
1. This synthesis is outlined in Fig. 2. Weigh 10.4 g of farnesyl acetone and 500 mg 10% Pd
on charcoal, dissolve in 100 mL methanol and hydrogenate at 50 psi in a Parr hydrogenator
for 2 d. Analysis by TLC (7:3 hexanes/diethyl ether) and GC-MS should indicate that the
material is fully converted to the hexahydrofarnesyl acetone.
2. Filter the reaction mixture over filter paper, wash with ethanol, rotary-evaporate to dryness,
and take up in a small volume of ethanol, then filter over Fluorosil to remove the last
traces of catalyst. Concentrate the resultant oil to dryness with a rotary evaporator (yield
8.64 g, 81% recovery).
3. Dissolve 1.56 g hexahydrofarnesyl acetone in 10 g dry dimethylformamide in a roundbottom
flask submerged in ice water and equipped with a dropping funnel containing
1.37 mL triethyl phosphonoacetone. Flush the entire apparatus with argon (Ar). Add
triethyl phosphonoacetone over the course of 45 min, followed by 2.2 mL 21% (w/w)
sodium ethoxide in ethanol, which is added over the course of 1 h.
4. Remove the mixture from the ice water bath and continue to stir for 48 h under Ar. Analysis
by TLC (7:3 hexanes/diethyl ether) should show that the majority of the starting material
(Rf 0.5) has reacted (final product Rf 0.6).
5. Transfer the reaction mixture to a separatory funnel with hexane, and wash 2× with a
NaCl-saturated solution. Dry the organic layer with MgS04, pass through filter paper, and
evaporate the solvent to dryness by rotary evaporation (yield 1.41 g, 72% recovery).
6. Dissolve 0.75 g of the product from the previous step in 10 mL anhydrous diethyl ether in
a round-bottom flask equipped with a stir bar. Cool the apparatus by stirring in an icewater
bath.
7. Add 267 mg of LiAlH4, and stir the reaction mixture overnight under Ar. The following
day, add 25 mL saturated NH4Cl and stir the reaction mixture overnight under Ar.