Page 1 \ ?^p 6r.1 CELL CYCLE CONTROL OF HUMAN H4 ...
Page 1 \ ?^p 6r.1 CELL CYCLE CONTROL OF HUMAN H4 ...
Page 1 \ ?^p 6r.1 CELL CYCLE CONTROL OF HUMAN H4 ...
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arE F: CeI Crct Cotuatat 114 G40 fa@ ptian<br />
by incubation lor io to 30 minutes at 24'C and elect ophoresis wh€reas<br />
H NF.M and HiNF-P prctein/DNA complexes were rosolved ln 4% (20r1) nal ve<br />
polyacrylamids gels using 1 t TGE Ois_base/Hcl Glvcine' EoTA PH 8.5)<br />
b!ffer (Ausub€l el a/., 1987). Bindlig readion mi{lres lor HiNF_D were<br />
elactrophoresed in 4% (8Ol acrylarnide/N,Nh€thvlenebisacrylamide weight<br />
relio) poyacrylamide gels in 0.5 x TBE bulf€r' Oel supershift assavs were<br />
carried out by p.einclbalion ol nuclear proteins (10 pl; 2_5 ,rg) wilh antibodv<br />
0 pD lor 15 min on ice, followed by additon of 10 r,l oi ONA mixtur€ and<br />
lncubation ior 15 min at room temperalLrrs pior 10 eleclrophores s<br />
Translenr ery.ession analys's<br />
wd rype <strong>H4</strong> promoler cAT constrlrcls (ie, Fo108_cAI) and ths<br />
analogous muianl promolsr cal construcls w€rs tansrecled in human H€La<br />
q cetuical carcinoma cells, .onaldiploid rat calvarial oslsoblasls (FoB)and<br />
ratosl€osarcoma 1712.8 (FOS) cells (S€€ Fiq.la) Helacels were cultured in<br />
Dulbeccos minimal €ss€ntial medium (DMEM) usinq high glucose<br />
concentrations and a combination ol5% feta calt s€rlfr and selo donor hors€<br />
serum. Ihe medium was supplemented wilh antibiotics ad amino acids at lhe<br />
lndicated linal concentralonsr respeclively, 100 lnil/ml penicilln l00 rglrnl<br />
slreptomycin and 2 mM of L-gldamin€ Monolayer cuhlres were se€ded at a<br />
density of 1x1@ per 1oo mm lissue cullur€ p'ate in 10 ml of olvEM and were<br />
incubated at 37'C lnder atmospheric condi ons ot 5% co, The nert day<br />
ce|ls were translected lvilh plasmid DNAS or inle.esr'
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