Horseradish peroxidase hrp conjugated secondary antibody

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.

Cells or tumor tissue samples were lysed on ice for 30 min in RIPA buffer (Beyotime Biotechnology) containing 1% protease inhibitor cocktail and phosphatase inhibitor cocktail (Biotool, Houston, TX, USA). The lysates were then centrifuged at 12,000 g for 10 min at 4°C. The total protein concentrations were estimated using a BCA Protein Assay Kit (Solarbio Life Sciences, Beijing, China). Identical amounts of cell lysates were boiled for 10 min and then separated by SDS-PAGE. After electrophoresis, the proteins were transferred onto a nitro-cellulose membrane. The membrane was blocked with 5% non-fat milk in Tris-buffered saline and 0.05% Tween 20 (TBST) for 2 h at room temperature and then incubated with primary antibody overnight at 4°C. The membranes were washed three times with TBST and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Zhongshan Golden Bridge, Beijing, China) for 2 h at room temperature. The membranes were then washed three times with TBST and immunoreactive bands visualized using enhanced chemiluminescence (ECL) reagent (Pierce Fast Western Blot Kit, Thermo Scientific, Waltham, MA, USA) according to the manufacturer's protocol. The relative expression ratios for the experimental and control groups were calculated based on density using the ImageJ software and the GAPDH signal as a reference.

Cells were lysed with RIPA buffer (Sigma-Aldrich, St. Louis, MO) supplemented with fresh protease inhibitor phenylmethanesulfonyl fluoride on ice. Protein concentrations were measured using a BCA Protein Assay Kit (Pierce Biotechnology Inc., Rockford, IL, USA). Protein (50 μg) was boiled and loaded on 10 % sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a PVDF membrane (Millipore, Bioprocess Technology Center, Billerica, MA, USA). Membranes were blocked in 5 % non-fat milk and incubated overnight at 4 °C with the appropriate primary antibodies at dilutions specified by the manufacturer. Antibodies against SIGIRR, phospho-IκBα (p-IκBα), phospho-ERK1/2 (p-ERK1/2), phospho-JNK (p-JNK), phospho-p38 (p-p38), p-38 and β-actin were purchased from Santa Cruz Biotechnology (California, USA). They were next washed three times in 15 ml Tris-buffered saline with Tween 20 (TBS-T) and incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (Zhongshan Golden Bridge Biotechnology, Beijing, China) at 1:1000 dilution in TBS-T for 2 h at room temperature with slightly shaking. Then the bound secondary antibodies were detected using ECL Western blotting Detection System (Amersham, Uppsala, Sweden).

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).

Total protein was lysed in radioimmunoprecipitation assay (RIPA) buffer (Highly Efficient Solitaire, Solarbio, China) containing 100 μM of phenylmethylsulfonyl fluoride (PMSF) (Solarbio, China) and protease inhibitor cocktail (Abcam, Waltham, MA, USA). Proteins measuring 25 μg were loaded and separated into 4%–12% NuPAGE gel (Beyotime, Shanghai, China), and they were transferred to 0.2 μm of nitrocellulose membranes (Millipore, Billerica, MA, USA). Then 0.1% Tween-20 and 5% BSA were provided to block the membranes for 1 h, followed by overnight incubation with primary antibodies (1:1,000). The membranes were washed and incubated for 1 h in horseradish peroxidase (HRP)-conjugated secondary antibody at 1:5,000 (Zhongshan Golden Bridge, China). Super Signal West Pico Chemiluminescent Substrate (Thermo) and BioMax film (Kodak, Rochester, New York, USA) were used to detect the immunoreactive proteins.

Both HepG2 and Huh7 cells were transfected with control miRNA or miR-505 mimics. After transfection for 48 h, cells were harvested and lyzed with the RIPA buffer (Beyotime, Shanghai, China) on ice for 10 min. The protein concentration was determined via the BCA protein assay (Beyotime, Shanghai, China). Equal amount of protein was separated by SDS/PAGE and transferred on to the nitrocellulose membranes (Millipore, Bedford, MA, U.S.A.). After blocking with 5% non-fat milk for 1 h at RT, membrane was incubated with the primary antibody (anti-IGF-1R (9750S), anti-pAKT (Ser⁴⁷³, 9271S), anti-AKT (9272S), Cell Signaling Technology, Danvers, MA, U.S.A.; anti-GLUT1 (sc-377228), Santa Cruz Biotechnology (Dallas, TX, U.S.A.) for 2 h at RT. Afterward, membrane was incubated with horseradish peroxidase (HRP)–conjugated secondary antibody (1:3000, Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) for 1 h at RT. Protein bands were visualized with Enhanced ECL detection kit (Millipore, Bedford, MA, U.S.A.).