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

Azi' F. Cet Crcl. C.nttat ot H4 G.n6 fans.4ttoh
pepstarrn,4psin inhibiror, TPoK, EoTA, and €GTA Bo€hringer, Indianapotis,
rN) Also, desaring was nor penormed by diatysis btjl by ditdion wi|h srorage
buffer wthout KCI (2 gtycerot, 0.2 mM EDTA, O.O1% Np4O, 25 mM H€pes,
pH7.5 and 1 mM DTT; kNO b!ff€4, Att€.nativ€ty, samplEs w6rs desalted by
gel tihration lsing Po-10 cotumns (p-! Biochemicats), or uttrafillraion using
c€nr.iconro unirs (amicon, Lenngton, MA)_ Finat protein conc€ntraions were
6dlusred wilh storage bufier {KNo bufi3r wiih 1clo mM Kct = KNlm bufie4
Prorein.DNA bindlng reactions ior deleciing BiNF.D were caiiBd our by
combining 10 rl ol a prot€in mixturo (in KNICTO storage boltet 20% gtyoerc,
100 mM KC1,0.2 mM EOTA, 0.01% Nonidst (N) p-40, 1 mM dithiothreitot, 25
mM HEPES lN,2-rlyd.otyerhytpiperaine-N:2-ehanesu onic acidl NaOH, pH
7.5)wrh 10 rror a DNA mirllre contaihing 1 plot probs oNA 0o tmotin TE
bun€4, 2 rr poly(dc.dc)i(dc-dc) (2!g in TE bufier), and 1 pt of pory(dt-
dclt{dl-dc) {0 2 !g in TE bufie4. Atternartvety, binding reacrions io. delectiog
HiNF-P/HiNF-M wer6 prepared by mixing 10 ,rl ot a p,orei. sotulion (in
KN100/MZ b!tle., whlchis ike KNICrO bur inctudes O.ZmM MgCt2andO.2mM
ZnCl2)with 10 plof a DNA mxrure containing 1 /l ot prob6 DNA (10 fmotjnTE
bule4, 1 ! ol salmon sperm DNA (Sigma) (2 /rg in TE bufis4, and 1 !t ot a
divalg cation soluiion (conraining 2 mM MgCt, and 2 mM ZnCt,).
competirion analyses wer€ pertorm€d by mixing 10 |lt ot DNA mtxture
conlanng 10 imol ol probe and 1,0oo tnot (,1e., 1OO iotd motff 6xcess) or
lnrabe|ed specific compeiiror doubte-srr€nd€d DNA with 10 !t ot protein
mi{ue conlaining approximately 1 ro 10 pg ot Heta nuct€ar prorein, tolowed
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