PMC:2632902 / 7262-8902 JSONTXT

{"project":"2_test","denotations":[{"id":"19043076-11875063-76939174","span":{"begin":274,"end":276},"obj":"11875063"},{"id":"19043076-15788687-76939175","span":{"begin":681,"end":683},"obj":"15788687"}],"text":"Transfection with targeted si-RNAs\nA 21-nucleotide duplex si-RNA (si-CKIδ/ɛ) with the sequence sense: 5′-CUGGGGAAGAAGGGCAACCdTdT-3′ and antisense: 5′-GGUUGCCCUUCUUCCCCAGdTdT-3′, purchased from Qiagen, Valencia, CA, USA was used to target identical regions in CKIδ and CKIɛ (29). In addition the On-Target plus SMART pool si-RNA anti-CSNKID human (si-CKIδ) and On-Target plus SMART pool si-RNA anti-CSNKIE human (si-CKIɛ) were purchased from Dharmacon, Lafayette, CO. For targeting CKIIα RNA the siGENOM SMART pool CSNK2A1 (Dharmacon, Lafayette, CO) was employed. The CKIIα′ si-RNA (sense: 5′-CAGUCUGAGGAGCCGCGAGdTdT-3′, antisense: 5′-CGGCUCCUCAGACUGdTdT-3′), previously described (30) was synthesized by MWG Biotech, Ebersberg, Germany. The control si-RNA (5′-GCUCAGAUCAAUACGGAGAdT dT-3′) was purchased from Dharmacon, Lafayette, CO. HCT-116 cells were incubated in serum-free McCoy's medium for 6–10 h with the si-RNA (100 nM) in the presence of Lipofectamine 2000 (Invitrogen Life Technology, Carlsbad, CA) as described by the manufacturer. When the combination of si-CKIδ and si-CKIɛ was employed, the concentration of each si-RNA was reduced to 75 nM. Following the initial incubation, cells were washed and cultured in McCoy's medium containing 10% fetal bovine serum and 2 mM l-glutamine for 24 h. At the end of the incubation period, cells were harvested for preparing cell lysates. When cells were transfected with si-CKIδ/ɛ about 50–70% of the cells that readily detached upon washing were used for preparing lysates for 2D-phosphopeptide maps of topo IIα, since both CKIδ and CKIɛ were maximally down-regulated in this population."}