Kif11

The transfected cells were cultured for 48 h and then collected for Western blot assay. The cells were lysed with radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific) at 4°C for 10 min. Then, protein concentration was quantified by the BCA Protein Analysis Kit (Beyotime, China). SDS-PAGE was utilized to electrophorese equal amounts of protein samples. Then, proteins were transferred to a PVDF membrane. At room temperature, the membrane was sealed for 1 h with 5% skimmed milk diluted with TBST. Then, primary antibody KIF11 (Abcam) or GAPDH (Abcam) was added, and the membrane was incubated overnight at 4°C. The membrane was fully rinsed with TBST, followed by hybridizing with goat anti-rabbit IgG antibody conjugated with horseradish peroxidase (Abcam, Cambridge, UK) for 2 h. After that, TBST was utilized to rinse the membrane three times. Finally, luminescence was carried out with an enhanced chemiluminescence (ECL) kit (Solarbio, China). All protein bands were observed by photo taking.

All proteins from cells or tumors were resolved by SDS-PAGE gel (P1200, Solarbio, Beijing, China) and transferred onto PVDF membranes (LC2002, Thermo, Waltham, US). Then, the membranes were blocked in 5% fat-free milk in TBST for 1 hour at room temperature and then washed, and primary antibodies were incubated at 4°C overnight. After being washed, the PVDF was incubated with horseradish peroxidase-conjugated secondary antibodies. The bands were visualized by ECL reagent (32106, Thermo). Antibodies are as follows: KIF11 (1 : 1000 dilution, ab61199, Abcam, Cambridge, UK); anti-β-actin (1 : 1000 dilution, ab5694, Abcam, Cambridge, UK).

Acetylated α-tubulin (Sigma-Aldrich, St. Louis, MO, USA, #T7451), tyrosylated α-tubulin (Genetex, Irvine, CA, USA, #GTX76511), β-tubulin (Sigma-Aldrich, #T5201), KIF11 (Abcam, Cambridge, MA, USA, #ab51976), vinculin (Sigma-Aldrich, #V9131), pTyr397-PTK2 (Thermo Fisher Scientific, Waltham, MA, USA #700255), α-adaptin (Thermo Fisher Scientific, #MA1-064), γ-H2AX (Genetex, #GTX127340; Cell Signaling Technology, Danvers, MA, USA, #9718), pSer345-CHK1 (Cell Signaling Technology, #2348), pThr68-CHK2 (Cell Signaling Technology, #2197), and NDC80 (Abcam, #ab3613).