Super rx film

iDCs were pre-incubated for 1 h with SP600125 and SB203580 (20 μM), and then infected with EV71 at a MOI of 5 in the presence of SP600125 and SB203580 for 24 h. Cells were harvested by centrifugation, washed and lysed with a lysis buffer (2% sodium dodecyl sulfate, 35 mM β-mercaptoethanol, 50 mM Tris–HCl (pH 6.8), 1 mM phenylmethylsulfonylfluoride). Cell lysates were obtained by centrifugation at 45,000 × g for 1 h at 4°C. Total protein concentration was determined by the bicinchoninic acid protein assay kit (Pierce). Equal amount of proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto PVDF membranes (Millipore). The membranes were blocked for 2 h with 5% nonfat dry milk solution in Tris-buffered saline containing 0.1% Tween-20 and then incubated with specific primary antibodies. After washed with PBS, the membranes were incubated with HRP conjugated secondary antibodies and washed with PBS. The immunoreactive bands were detected by ECL reagents (GE Healthcare), visualized on Super RX film (Fujifilm) and quantitated by densitometric analysis (ImageQuant, Molecular Dynamics and PDSI, GE Healthcare). The level of phosphoproteins was normalized to its respective control at 0 h, which was arbitrarily set to 1.

The protein content of cell lysates was determined by Bradford analysis, and approximately 20 μg of total protein was used for each sample. Protein samples were resolved by SDS-PAGE and electrophoretically transferred onto nitrocellulose membranes. After blocking in 3% milk for 60 min, membranes were incubated with primary antibody at 4 °C overnight. The primary antibodies were detected with their corresponding horseradish peroxidase-conjugated secondary antibodies followed by enhanced chemiluminescence development (Pierce Chemical, Rockford, IL, USA) and light detection with Fuji (Tokyo, Japan) Super RX film.

MCF7 cells transfected with different miRNAs were lysed in RIPA buffer. Protein concentration was detected were determined, loaded on 10% SDS-polyacrylamide gels and transferred to PVDF membranes. Then the PVDF membrane were incubated with anti GRM4 antibody (ab53088, Abcam) at 4 °C overnight and subsequently incubated with an HRP-conjugated anti-rabbit antibody for 1 h. The blots were visualized by ECL methods using FUJI SUPER RX film.

iDCs were pre-incubated for 1 h with U0126 at different concentrations as indicated and then infected with EV71 at a MOI of 5 in the presence of U0126 for 48 h. Cells were harvested by centrifugation, washed and lysed with 50 mM Tris pH 6.8, containing 250 mM NaCl, 2% sodium deoxycholate, 0.5% NP-40 and 1 mM PMSF. After vortexing and incubating on ice for 10 min, cell lysates were centrifuged at 10,000 × g for 5 min. Total protein concentration was determined by the bicinchoninic acid protein assay kit (Pierce). Proteins in the lysates were separated by sodium dodecyl sulfate polyacrylamide-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred to PVDF membranes (Millipore), and probed with specific primary antibodies. After washed with PBS, the membranes were incubated with horseradish peroxidase conjugated secondary antibodies. The specific proteins were detected by ECL reagents (GE Healthcare), visualized on Super RX film (Fujifilm) and quantitated by densitometric analysis (ImageQuant, Molecular Dynamics and PDSI, GE Healthcare). The relative amounts of the phospho-ERK1/2 (p-ERK1/2) were normalized to GAPDH after densitometry scanning and analysis with the band leader software (version 3.0).

Total protein was extracted and the concentration was determined by BCA assay. Twenty microliters of the sample was loaded into each well for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a PVDF membrane (Millipore, Billerica, MA, USA). The membrane was blocked for 2 h with 5% nonfat dry milk solution in TBST (Tris-buffered saline containing 0.1% Tween-20) at room temperature and incubated with primary antibodies (VP1, 1:500; 2A, 1:1000; 3C, 1:1000; and 3D, 1:500) overnight at 4 °C, and followed by incubation with secondary antibody conjugated with horseradish peroxidase (Proteintech) for 1 h. The immunoreactive bands were detected by Super RX film (Fujifilm, Tokyo, Japan) and quantified by ImageQuant densitometric analysis (Molecular Dynamics, Sunnyvale, CA, USA).

Protein samples were separated by SDS-PAGE and blotted on PVDF (polyvinylidenfluoride) membranes (Merck Millipore). Membranes were blocked for 1 hour with Tris-buffered saline, 0.05% Tween 20 (TBST), and 5% dry milk and then incubated for an additional 1 hour with the primary antibodies diluted in TBST, 5% dry milk. The membranes were washed 4 times for 5 minutes in TBST and incubated for 1 hour with the secondary antibody diluted in TBST, and 5% dry milk. The membranes were finally washed again 4 times for 5 minutes in TBST and revealed with Immobilon Western Blotting Chemoluminescence HRP substrate (Merck Millipore) and Super RX-film (Fujifilm). Rabbit antisera were used at the following dilutions: anti-CtrA (1:10,000), anti-PilA (1:10,000), anti-FljK (1:50,000), anti-CpaC (1:5000), anti-ZitP (1:5000), anti-CpaM (1:5000) and anti-GcrA (1:2000). HRP-conjugated Anti-rabbit secondary antibody was used at 1:20,000 dilution (Jackson ImmunoResearch, USA).