Apolyvinylidene fluoride membrane

Western blotting was performed as described previously.^{13 (link)} Briefly, proteins were extracted from previously frozen cancer and
paracancer tissues by lysing them in radioimmunoprecipitation assay buffer
containing complete protease inhibitor cocktail (Pierce Chemical Company,
Rockford, IL, USA). The protein concentration was determined with the Bradford
assay (Bio-Rad, Hercules, CA, USA). Denatured protein (200 μg) was separated by
sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a
polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). The membrane
was blocked with 5% nonfat milk in Tris-buffered saline–Tween 20 for 2 hours,
then incubated with rabbit anti-vinculin (1:800; Abcam, Cambridge, MA, USA) and
rabbit anti-β-actin antibody (1:1000; Abcam) at room temperature for 12 hours.
The membranes were incubated with horseradish peroxidase (HRP)-conjugated goat
anti-rabbit IgG secondary antibody (1:2000; Abcam) for 2 hours after washing
with rinse buffer at room temperature. Membranes were developed with enhanced
chemiluminescence (Pierce Protein Research Products), and optical densities were
analyzed by ImageMasterTM2D Platinum (Version 5.0; Amersham Pharmacia Biotech
GE, Shanghai, China).

BMMCs were seeded into a 6-well plate (1 × 10⁷ cells/well) and then
sensitized with DNP-IgE for 24 h. Cells were then incubated with different
concentrations of LicA for 30 min followed by HSA for stimulation for 30 min,
and collection of total proteins using RIPA lysis buffer containing 10% protease
inhibitor (Roche Diagnostics) and phosphatase inhibitor cocktail (Roche
Diagnostics) in ice-cold conditions. The protein concentration was determined
using Bio-Rad protein analysis reagent. Proteins were dissolved in 5× loading
sample buffer and boiled for 5 min, then equal quantities of protein were
separated using SDS-PAGE. The proteins were then transferred onto a
polyvinylidene fluoride membrane (Millipore, MA, USA; HVLP04700), then blocked
in a blocking solution (5% skim milk) for 1 h at 37°C, followed by incubation
with primary antibody (1:1000) for 12 h at 4°C, and washed with Tween 20/Tris
buffered saline (TBST) three times. The secondary antibody (1:20000) conjugated
with horseradish peroxidase was incubated for 2 h at room temperature. The blot
was washed three times with TBST and then visualized with an enhanced
chemiluminescence (ECL) kit. The images of the developed blots were captured by
a Lane 1DTM transilluminator (Beijing Creation Science Co., Ltd., Beijing,
China) and were analyzed by Image-Pro Plus 5.1 software (Media Cybernetics,
Inc., Rockville, MD, USA).

Cells lysate was prepared using T-PER Tissue Protein Extraction Reagent (Thermo
Scientific) supplemented with Halt™ phosphatase and protease
inhibitor cocktail (100X) (Thermo Scientific). Proteins were separated on sodium
dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a
polyvinylidene fluoride membrane (Millipore). After blocking with 5% skim
milk, the membrane was incubated with primary antibodies, followed by incubation
with horseradish peroxidase-conjugated secondary antibody. Target proteins were
visualized using Super Signal West Pico Chemiluminescent Substrate and
ImageQuant 800 (Amersham).