Ab37150

PBS was used to wash fresh tissue which was grounded into a homogenate, and then RIPA buffer was used to lyse them to obtain total protein. Protein concentrations were determined by the BCA assay (Solarbio, PC0020, China). Samples (30 μg protein) were resolved on 8% SDS-PAGE gel and transferred to PVDF at 4°C. Membranes were blocked in fat-free milk combined with TBST for 1 hour at 25°C and immunoblotted with the appropriate diluted primary antibody KIF20A (Abcam, ab104118, UK; 1 : 1000 dilution for Western blot and 1 : 200 dilution for IHC-P), PCNA (Abcam, ab92552, UK; 1 : 1000 dilution for Western blot), Ki67 (Abcam, ab16667, UK; 1 : 1000 dilution for Western blot), MMP2 (Abcam, ab37150, UK; 1 : 500 dilution for Western blot), MMP9 (Abcam, ab76003, UK; 1 : 5000 dilution for Western blot), and GAPDH (SunGene Biotech, KM9002, China; 1 : 5000 dilution for Western blot) at 4°C overnight. Then, membranes were washed 10 minutes three times and incubated with the secondary antibody for 1 hour at 25°C. Finally, the blots were visualized by Imager.

Total cell lysate was extracted using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology). Approximately 30 µg of protein was separated by 10% SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% skimmed milk, the membranes were incubated overnight with primary antibodies against CEP55 (Abcam, ab170414), MMP2 (Abcam, ab37150), MMP9 (Abcam, ab38898), Cyclin D1 (Abcam, ab134175), p-IKKβ (Abcam, ab59195), IKKβ (Abcam, ab32135) and IκBα (Abcam, ab32518), followed by incubation with secondary antibodies. Human α-tubulin (Abcam, ab7291) or GAPDH (Abcam, ab8245) were used as the endogenous references, according to the details. Immunoreactive protein bands were visualised using the ECL method (Invitrogen), according to the manufacturer’s recommendations.

Cells were lysed with a RIPA buffer (CW2333S, CWbio, China) and a protease inhibitor cocktail set I (539131, Millipore, USA). The cellular proteins' supernatants were collected by centrifugation at 14,000 g for 20 min at 4°C. Protein concentrations were measured with the BCA protein assay kit (CW0014S, CWbio). Equal amounts of proteins were separated by 10% SDS-PAGE (P0014B, CWbio) and were transferred to PVDF membranes (ISEQ00010, Millipore, USA), which were blocked for 1 h with 5% bovine serum albumin (0332, Amresco) and were incubated overnight at 4°C with related primary antibodies, including LDHA (1:1,000, C4B5, Cell Signaling) matrix metalloprotein 2 (MMP2, 1:1,000, ab37150, Abcam), ZO1 (TJP1 tight junction protein 1; 1:1,000, D7D12, Cell Signaling), E-cadherin (1:1,000, 24E10, Cell Signaling), N-cadherin (1:1,000, D4R1H, Cell Signaling), Vimentin (1:1,000, D21H3, Cell Signaling), Slug (1:1,000, C19G7, Cell Signaling), and β-ACTIN (1:1,000, D6A8, Cell Signaling). After probing with secondary antibodies (GK600510">GK600510, Gene Tech, China), the membranes were conjugated to horseradish peroxidase (HRP) in room temperature for 1 h, and the bands were exposed by a chemiluminescent HRP substrate (WBKLS0500, Millipore, USA). The quantitative analysis of Western blots was carried out by Image J (https://imagej.nih.gov/ij/download.html).