Cell lysates were prepared and subjected to immunoblot analysis of Plk3 protein. Cells (about 1×107 cells) washed twice with ice-cold PBS were lysed with RIPA lysis buffer (no. P0013B; Beyotime Biotechnology, Shanghai, China) and complete protease inhibitor (cocktail, no. B14001a; Selleck, Shanghai, China) for 30 minutes on ice and then cleared by centrifugation at 12,000 rpm at 4°C for another 30 minutes. The total protein concentration in the extracts was measured utilizing a bicinchoninic acid protein assay kit (Beyotime Biotechnology). Equal amounts of protein were separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% BSA or non-fat dry milk in a mixture tris-buffered saline (TBS) and Tween 20 for 1 hour and then probed with antibodies against Plk3 (D14F12, Rabbit mAb, 1:1,000; Cell Signaling Technology Danvers, MA, USA), β-tubulin (abs830032, 1:1,000; Absin, Shanghai, China), GAPDH (abs130609, 1:1,000; Absin), SLUG (sc-166476, 1:500; Santa Cruz Biotechnology Inc., Dallas, TX, USA), Claudin-1 (D3H7C, 1:1,000; Cell Signaling Technology and D3H7C, American) and MMP2 (sc-13594, 1:500; Santa Cruz Biotechnology Inc.). Western blotting was performed as previously described.20 (link)