Page 1 \ ?^p 6r.1 CELL CYCLE CONTROL OF HUMAN H4 ...

Azh F: C.tt cycla cdt@t al H4 c.@ ftansipn@
Itatetuts dnd Mer4ads
The CAT reaciions were pedorm€d in a Inal volume of 150 pl
coniaining lcro - 250 !g tola prolein in97.5 ploi €xtract, io llloll lv T'is/HCl
(pH 7.8),2.s !lolrrc ch oramphonlcol0 25 roi(AmeGham) anda0'rlol39
mg/ml acelylcoenzyme A (Sigma)(final conceniration: 4 mM) B€adions wers
incubated lof 1 to 3 houG depending on the level ol cAT activlty in the
samples, The approximate levsl oi CAT sctivily was determined bv pre-
evaluating a subset ol ihe samples bv slandard reaclio's io establish that lhe
CAT assay conditions remain in lhe linear r6nge (e q'' 510 10 rl oi HeLa cell
tsate to. t hr, 30 ril ol FOS and ROB ceLl lysaie lor 3 hr) The rcact ons were
stopped by adding 1 ml oi cold €lhylaceiate ssmples were vortexed ior 30
seconds to mix lh6 organic phas6 wit'h the aqu€ous phase The phasss w6rs
separaied by briel centrilugation and th6 organic phase conraning lhe
chLorornphenlcaL prodLrds was iranslercd io enolher lube The orqanic
solvenl was allowBd rc evaporats over'ight und€r lh6 hood or dried using a
Speed vac apparatls The samples wer€ rcsuspended in 20 _ 25 pl ol
ethylacetate and loaded onlo a silica gel thin laye' chromatographic FLc)
plale. Plates wers placed in a mixlur€ ot equilibrated chloroiorrn:methanol
(9s:5 V/v) in an ascending develoPing tank and lhe samples were run lor 45
minlrtes. Ihen plates were air d ed ior 30 soconds und" |he hood and were
exposed lo XOMAT AR Fih ovehighl at room tenperature wilhout
intsnsiting screen. caT acriviiv on lh6 TLC plates was determined bv placing
rhe plalss inthe Belascope 603 analyzgl (Bstagen) ior30minutes The levelot
erpressiof lor each mutant cAT construcl was evauated as percent
convers on r€laliv€ to thewildlvp€ construcl'
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