Western blot analysis

Cells were lysed with RIPA buffer [25 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X 100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, protease inhibitor cocktail (Roche, Indianapolis, Indiana), phosphatase inhibitor cocktail (Roche)]. Lysates were quantified using the Pierce BCA Kit (Thermo Scientific), and equal amounts of protein were denatured and loaded onto an 8% SDS-PAGE gel. The protein was transferred onto a polyvinylidene difluoride membrane using the Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, California). The membrane was blocked in 5% non-fat milk-TBST and then incubated with anti-MELK (abcam, Cambridge, MA; ab108529) at a 1:3000 dilution, or blocked in 10% BSA-TBST and then incubated with anti-MELK (Cell Signal, Danvers, MA; 2274S) at a 1:1000 dilution. Anti-alpha-tubulin (Sigma-Aldrich, St. Louis, MO; T6199) was used as a loading control at a 1:3000 dilution. All primary antibody incubations were performed overnight at 4°C. Following incubation, the membranes were washed and then incubated in either anti-rabbit secondary (abcam; ab6721) at 1:50000 for MELK or anti-mouse secondary (Bio-Rad; 1706516) at 1:10000 for Tubulin for 1 hr at room temperature.