Enhanced chemiluminescence ecl system

Protein samples were separated by electrophoresis on 10% (w/v) SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad). The membranes were then blotted with corresponding antibodies. Subsequently, the membranes were washed four times with TBST (0.05% tween-20 in Tris-buffered Saline) and incubated with horseradish peroxidase (HRP) conjugated goat-anti-mouse (Sigma-Aldrich) or goat-anti-rabbit IgG (Sigma-Aldrich). The enhanced chemiluminescence (ECL) system (Thermo) was utilized to detect the blotted proteins.

Prepared HUVECs were homogenized with RIPA buffer (Thermo Fisher Scientific) and resolved by SDS-PAGE. To detect secreted PAI-1, conditioned media was collected, mixed with 5× sample buffer, and boiled for Western blot analysis. The protein content was normalized by Bradford assay. Prepared cells were resolved by SDS-PAGE, and transferred to PVDF membranes (Millipore, USA). After transfer, the membranes were blocked with 5% skim milk in TBS-T (10 mM Tris/HCl, pH 8.0, 150 mM NaCl, 0.05% Tween-20) for 1 h, and incubated with the specific primary antibody in the blocking solution. Western blotting was performed with specific antibodies for PAI-1 (BD bioscience), phospho-JNK, JNK, phospho-p38, p38 (Cell signaling technology), and β-actin (Santa Cruz Biotechnology). The membranes were incubated with horse-radish peroxidase-conjugated secondary antibody in TBS-T followed by chemiluminescence detection using an enhanced chemiluminescence (ECL) system (Thermo Fisher Scientific, USA).

RIPA was used for protein extraction (Beytime, Shanghai, China). Proteins were isolated with 10% SDS/PAGE, transferred on to nitrocellulose membranes and blocked for 1 h by TBS containing 5% skim milk. Primary antibodies were used for incubation of members overnight at 4°C, including anti-TNS1 (1:1000, ab167660, Abcam), anti-E-cadherin (1:2000, SC-71008, Santa Cruz Biotechnology), anti-N-cadherin (1:2000, SC-59987, Santa Cruz Biotechnology), anti-vimentin (1:2000, SC-32322, Santa Cruz Biotechnology), anti-RhoA (1:2000, SC-418, Santa Cruz Biotechnology), anti-Akt (1:2000, SC-56878, Santa Cruz Biotechnology), anti-phospho-Akt (1:2000, SC-377556, Santa Cruz Biotechnology) and GAPDH (1:800, 5174, Cell Signaling Technology). Then, members were maintained with secondary antibodies at room temperature for 1 h. An enhanced chemiluminescence (ECL) system (Pierce Biotechnology, Rockford, U.S.A.) was recruited to visualize the bound antibodies. GAPDH was employed as a loading control.

Equivalent quantities (30–50 µg) of protein were separated by 10% SDS polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with 5% non-fat dried milk and incubated overnight with the appropriate primary antibody at dilutions specified by the manufacturer. Next, the membranes were washed three times in TBST and incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody at 1∶5,0000 dilution for 1 h. Bound secondary antibody was detected using the Enhanced Chemiluminescence (ECL) system (Pierce Biotechnology Inc, USA). Primary immunoblotting antibodies were: anti-GAPDH, anti-PAQR3 (Santa Cruz Biotechnology, USA).

Briefly, cells were harvested at 48 or 72 hours following dsRNAs treatment as described above, washed and lysed with lysis buffer. Protein concentration in the resulting lysate was determined using the bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, IL, USA). Appropriate amounts of protein (30-50 µg) were resolved by electrophoresis in 10-15% Tris-glycine polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked and then incubated overnight with the appropriate primary antibody at dilutions specified by the manufacturer. Primary immunoblotting antibodies were purchased from the Cell Signaling Technology, Beverly, MA, USA. The membranes were next washed three times in 15 ml TBST and incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody at 1:1000 dilution in TBST for 1 hour. After washing three times for 5 minutes each with 15 ml TBST, bound secondary antibody was detected using an enhanced chemiluminescence (ECL) system (Pierce Biotechnology Inc., Rockford, IL, USA).
To determine NF-κB cellular localization, nuclear and cytoplasmic proteins were isolated from the cells using a cell fractionation kit (KeyGen, Wuhan, China). NF-κB expression in the nuclear and cytoplasmic compartments was determined by immunoblot analysis as described above.