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

Aziz F: Celt Cyct. Contd d H4 6@e lanscttOtbn
chloramphenicol acelynransteras€ (cAT) assay
The CAT assay was perlormed as des.ribed bv Go'man er a/ 0982)'
C€lls were washed wilh 1 x PBS twic€ and wet6 s'raPed with a rubbe'
policeman in I ml ot TEs bufier (40 mM Tris l nM EOTA, 150 mM NaCl pH
7.5). cells ca. be easit peeled ofi nom rhe plate, €sp€cially FOS and ROB
cells by using TES bufior and tansl€ded in 1 5 ml c€ntrdug€ tub€ k€pt on rce
Th6 tubes sere cenlitlg€d lof 5 minul€s at 4 C th€ super'atant was
dis€fded and lh6 pellel was saved Subs€quentlv lOO rlol 0 25 M Tris' pfl
7.8 was added, ihe pelletwas resusp€nded w6ll and the suspension paced n
an ethanol drynce barh lo. 5 minules Th€ samPles wer6 ih6n tansre(od rc a
37 c watedalh ior 5 minul€s' Thls p'oc€du'6 was ropsaled iwo times (i € ' a
total oi lhree lre€z6thaw c-ycles) Th€ sampl€s w€re th€n centriflged lor 10
minltes al iul speed a.d the supernalani sav€d lor the acetvlalion reaction A
smallalqlol ot 6ach samplg (2 }ll) was sav6d lorlhs dotBrrninallon of prol€ln-
cofce.lratio.
Prolein-concentrations w€re dBterminod as lollows A s|andard'cldo
was obiained bv d€lermi.ing th6 absorbanco r€adings ol a saries ol saFpr6s
wirh known protei. co.ce.l'ations betwe€n 1 T g to 7 rr9 ot bovine serun
albumin (BsA, $andard slock 1 mg/ml) Aliquots (2 pl) ot each sample were
mded wilh I ml ol 1x coommassie prol€in assav teagont (Pierce) (obtained
by dillring lhe slock soldon2lold wnh dd H2O) and the opii':3ldensilv ol the
mi{ure at 595 nm was detemined The absorbancs values ol lhe iesl
samptes was compared wllh ihose ol ih€ BSA srandatd and corlespondlng
pro€in concedratonswerecalculaled
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