1 vector Expression plasmid for dominant negative mutant

1 vector. Expression plasmid for dominant negative mutant MK5108 of EGFR (EGFR-DN) had a deletion of 533 amino acids at the N terminus, which competitively inhibited the activation of EGFR, and was cloned into pcDNA3.1. The pSG5-STAT3 was obtained from whole STAT3 coding fragment cloned into XhoI sites of the pSG5 vector. Expression plasmid for dominant negative mutant of STAT3 (STAT3β) had a deletion of 55-residue in C-terminal transactivation domain of STAT3 and replaced by seven unique C-terminal residues (CT7) [44]. The EGFR and STAT3 motif mutation

(designated as pD1-mut-Luc) from pCCD1-Luc were generated by PCR based on an overlap extension technique. The primers used for generating mutations were: 5′- CTCCACCTCACCCCCTAAAT-3′ and 5′-AGGGATGGCTTTTGGGCTCT -3′. PCR-amplified fragments carrying the desired mutations were then cloned into Xba I sites of the pBSK + vector. The construction of expected TAKARA Biotechnology completed mutations and the sequencing of integrity of the vector. DNAzyme 1 (DZ1) is an LMP1-targeted DNAzyme that binds and cleaves LMP1 Sotrastaurin supplier RNA in a highly sequence-specific manner [19]. And the control oligonucleotide of DZ1 (TAKARA, China) was designed by inverting the catalytic core sequence. To monitor transfection efficiency, pRL-SV40 (Promega, U.S.A) was used as an internal control.

Preparation of cell lysates and cell fractions For whole cell lysates, 107/ml cultured cells were harvested and washed twice with ice-cold phosphate-buffered saline (PBS), and then lysed in the 500 μl lysis buffer [10 mM Tris–HCl, pH 8.0; 1 mM EDTA, 2% sodium dodecyl sulfate (SDS); 5 mM dithiothreitol (DTT); 10 mM phenylmethyl sulfonylfluoride (PMSF); 1 mM Na3VO4; 1 mM NaF; 10% (vol/vol) glycerol; protease inhibitors cocktail tablet (Roche,

Switzerland)] for 30 min on ice and centrifuged at 15,000 × g for 10 min. The supernatant was collected and stored at -70°C until used. For Preparation of cytoplasmic and (-)-p-Bromotetramisole Oxalate nuclear fractions, 107/ml cells were washed with PBS and suspended in 200 μl of lysis buffer (10 mM Hepes, pH 7.9; 10 mM KCl; 0.1 mM EDTA; 0.1 mM EGTA; 1 mM DTT; 0.5 mM PMSF; and protease inhibitor cocktail). The cells were incubated on ice for 15 min, after which 6.5 μl of 12.5% NP-40 was added; the contents were mixed and then centrifuged for 1 min at 12,000 rpm. The supernatant was saved as cytoplasmic fraction. The pellet was resuspended in 12.5 μl of ice-cold nuclear extraction buffer (20 mM Hepes, pH 7.9; 0.4 M NaCl; 1 mM EDTA; 1 mM EGTA; 1 mM DTT; 1 mM PMSF; and protease inhibitor cocktail) and incubated on ice for 40 min with mixing every 10 min, then they were centrifuged for 5 min at 12,000 rpm at 4°C. The supernatant was saved as nuclear fraction. The cytosolic and nuclear fractions were stored at -70°C until used.

Comments are closed.