Ecl plus chemiluminescence system

Mimics and inhibitors of miR-17-5p or miR-20a, cholesterol-conjugated miR-17-5p and miR-20a inhibitors, c-myc, uPAR, and DR5-specific small interfering RNA (siRNA) were chemically synthesized by RiboBio Co., Ltd. (Guangzhou, China). The sequence of siRNAs for c-myc, uPAR, and DR5 were as follows: c-myc, 5′-CTATGACCTCGACTACGAC-3′; uPAR, 5′-GCTGTACCCACTCAGAGAA-3′; DR4 5′-CUCUGAUGCUGUUCUUUGAtt and DR5, 5′-AAGACCCUUGUGCUCGUUGUC-3′. The following reagents and antibodies were obtained as indicated: DR5 antibody was from Santa Cruz Biotechnology (Dallas, TX,, USA); procaspase 8, caspase 3/8, and total and phosphorylated forms of ERK antibodies were from Cell Signaling Biotech; uPAR antibody was from R&D Systems (Minneapolis, MN, USA); and c-myc, actin, GAPDH, and horseradish peroxidase (HRP)-conjugated secondary antibodies were from Zhongshan Goldenbridge Biotechnology (Beijing, China). The ECL-Plus chemiluminescence system was from Applygen Technologies (Beijing, China).

Chemically synthesized c-myc- and C/EBPα-specific small interfering RNA (siRNA) and nonspecific control were purchased from RiboBio Co., Ltd. (Guangzhou, China). The sequences of the c-myc siRNAs were 5′-CTATGACCTCGACTACGAC-3′ and 5′-CTCGGTGCAGCCGTATTTCTA-3′, and the C/EBPα siRNA sequences were 5′-CCAAGAA GUCGGUGGACAADTDT-3′ and 5′-CGACGAGTTC CTGGCCGAC-3′. We purchased cholesterol-conjugated miR-122 mimic and negative control from Ribobio (Guangzhou, China) for RNA delivery in vivo. Reagents and antibodies were obtained as follows: hIL-6 (recombinant human Interleukin 6) and hTNFα (recombinant human tumor necrosis factor α) were purchased from Sinobio Biotechnology Co., Ltd. Shanghai China; DNase I (RNase-free) was purchased from Invitrogen; the Superscript RT reagent kit was from TaKaRa Bio Inc., Shiga, Japan; rabbit anti-human C/EBPα (sc-61 X) and mouse anti-c-myc (sc-40) monoclonal antibodies were from Santa Cruz Biotechnology; rabbit IgG was from Sigma; mouse anti-human actin, GAPDH and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Zhongshan Goldenbridge Biotechnology, China; the ECL-Plus chemiluminescence system was from Applygen Technologies, Beijing, China.