cycle they appeared to undergo apoptosis fig4 a 4d cells treated with dmso alone failed to exhibit such an arrest in the g2 m phase of the cell cycle suggesting that the effects seen are associated with fri 20 addition fig4 e 4h treatment of the normal cell lines nih3t3 or hfl with fri 20 failed to produce a similar effect on cell cycle progression nih3t3 and hfl exhibited normal cell cycle progression in the presence and absence of drug to examine the effects of fri 20 on the mapk pathway nih3t3 lncap and mcf 7 cells were incubated with fri 20 at a concentration 2 5 mm for 48 hours following incubation of cells in the presence and absence of fri 20 the cells were lysed using erk lysis buffer containing 20 mm hepes ph 7 4 50 mm u03b2 glycerophosphate 0 5 triton x 100 2 mm mgcl 2 1 mm egta 1 mm dithiothreitol 2 u03bcg ml leupeptin 2 u03bcg ml aprotinin 100 u03bcm phenylmethylsulfonyl fluoride and 1 mm benzamidine erk 2 in 100 mg of cell lysate was immunoprecipitated by incubating lysate protein with 1 mg of erk 2 polyclonal antibody antibody sc 154 to erk2 is from santa cruz biotechnology inc for one hour followed by an additional incubation of.