Immunoblotting

Cells were washed with PBS, and then lysed using RIPA buffer containing protease inhibitors (Sigma-Aldrich, MO, USA), phenylmethylsulfonyl fluoride, phosphatase inhibitors and dithiothreitol (Wako, Osaka, Japan). The protein concentration was measured using the DC protein assay (Bio-Rad, CA, USA). Cell lysates and conditioned media were electrophoresed on 10% SDS-PAGE gels and blotted onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). After blocking with 3% bovine serum albumin (Wako, Osaka, Japan) or 5% non-fat dry milk for 1 hour and washing 3 times with 1 × TBS containing 0.1% Tween (MP biomedicals, Illkirch, France) (TBST), the membranes were incubated with primary antibodies overnight at 4°C. The membranes were washed 3 times with TBST and incubated with a horseradish-conjugated secondary antibody for 1 hour at room temperature. After incubation, the membranes were washed 3 times with TBST and incubated with an enhanced chemiluminescence reagent (Millipore, MA, USA) for 5 minutes.