Cell lysate preparation and Western blot analysis

After stimulation, the medium was aspirated. Cells were rinsed twice with ice‐cold PBS, and 25–100 μl of cell lysis buffer (20 mM Tris–HCl, pH 7.5, 125 mM NaCl, 1% Triton X‐100, 1 mM MgCl₂, 25 mM β‐glycerophosphate, 50 mM NaF, 100 μM Na₃VO₄, 1 mM PMSF, 10 μg/ml leupeptin and 10 μg/ml aprotinin) was then added to each well. After harvesting, cell lysates were sonicated and centrifuged, and equal protein amounts of soluble protein, as determined by the Bradford protein assay, were denatured, subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS‐PAGE), and transferred to a polyvinylidene difluoride membrane. Non‐specific binding was blocked with TBST (50 mM Tris–HCl, pH 7.5, 150 mM NaCl and 0.02% Tween 20) containing 5% non‐fat milk for 1 h at room temperature. After immunoblotting with the first specific antibody, membranes were washed three times with TBST and incubated with a horseradish peroxidase (HRP)‐conjugated secondary antibody for 1 h. The dilution folds of first specific antibodies were 1:1000 and β‐actin was 1:10,000. After three washes with TBST, the protein bands were detected with enhanced chemiluminescence detection reagent. To make sure equal amounts of sample protein were applied for electrophoresis and immunoblotting, β‐actin was used as an internal control.