Semi dry transfer apparatus

After HCS-2/8 cells had been knocked down by the siRNAs against RFX-1, they were treated with NaF at the concentration of 5 mM for 16 h. Then, cell lysates were collected, and Western blot analysis was performed, as described previously (Sumiyoshi et al., 2013) . In brief, after protein concentrations of cell lysates had been determined by the Pierce TM BCA Protein Assay using bovine serum albumin (BSA) standards (Thermo Scientific, Rockford, IL, USA), each cell lysate containing an equal amount of proteins was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and was then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore) by using a semi-dry transfer apparatus (Atto Corp., Tokyo, Japan). The blot was reacted with anti-CCN3 (CST, Beverly, MA, USA; Cat # 8767S) and anti-β-actin (Sigma-Aldrich; Cat# A2228) antibodies overnight at 4 °C. After being washed with Tris-buffered saline (TBS)-Tween 20 (TBST) and TBS buffers, the blots were incubated for 60 min at room temperature in secondary antibodies conjugated with horseradish peroxidase (HRP). Subsequently, the membranes were washed with TBST and TBS buffers, and the bands were detected with the chemiluminescence substrate by using a LAS-4000 mini image analyzer (Fuji Film, Tokyo, Japan).