Ecl reagent kit

Cells and exosomes were lysed in Radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich, Oakville, ON. Canada) and quantified using the Bradford assay. Proteins were extracted by boiling for 5–10 min in Laemmli buffer (Biorad, Hercules, CA, USA) containing freshly added beta-mercaptoethanol (Sigma-Aldrich, Oakville, ON, Canada). Protein extracts were subjected to SDS-PAGE using Mini-PROTEAN precast gel 4%-15% (Biorad labs, Mississauga, ON, Canada), stained with Ponceau S and transferred to nitrocellulose membranes (0.45 µm, Amersham Protran). Membranes were blocked in 5% skim milk in TBST buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 0.02% Tween-20) for 1 h followed by incubation with rabbit monoclonal anti-CD9 antibody diluted 1:2000 in the same buffer (Abcam, Toronto, ON, Canada) and incubated overnight at 4 °C. Membranes were washed 3x in TBST and then incubated with goat anti-rabbit-HRP antibody diluted at 1:5000 in TBST (Sigma-Aldrich, Oakville, ON, Canada) for 1 h at room temperature. Membranes were washed 3x with TBST, and then HRP signal was detected using Enhanced Chemiluminescent (ECL) reagent kit (Boster, Pleasanton, CA, USA).

Oral mucosa lysate was used to examine the expressions of NF-κB. The protein concentration was determined by a Q5000 UV-vis Spectrophotometer. An equal amount of 20–30 μg of protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes by electroblotting equipment. Nonspecific protein binding was blocked with 1% bovine serum albumin in TBS with 0.1% Tween 20 (pH 7.6, 3.03 g Tris base, 18.8 g glycine, 1 g SDS, 1000 ml ddH₂O, plus 1 ml Tween-20) for 1 h at room temperature. Then, the membranes were incubated overnight at 4°C with the diluted primary antibodies. After washing with TBST three times, the membrane was incubated with the corresponding HRP-conjugated secondary antibody (1 : 2000) at room temperature for 1 hour and then washed for three more times with TBST. Protein bands were visualized using the enhanced chemiluminescence detection system (Boster ECL Reagent kit). The experiments were repeated three times independently. A monoclonal antibody specific to β-actin (1 : 500) was used to determine the equal protein loading.