Anti plk1

Cell pellets were resuspended in sample buffer, boiled and analyzed by western blotting. Membranes were probed with the following antibodies: anti-GFP (rabbit polyclonal, Abcam, ab190584, 1:10000), anti-Bub1 (rabbit polyclonal; Abcam, Cambridge, UK; 1:5000), anti-BubR1 (mouse monoclonal; BD #612503, 1:1000) and anti-Tubulin (mouse monoclonal; Sigma; 1:8000), anti-Incenp (rabbit polyclonal, Cell Signaling #2807, 1:500), anti-Cyclin B (mouse monoclonal, Santa Cruz, sc-245, 1:1000), anti-PLK1 (mouse monoclonal; Abcam #ab17057, 1:1000), anti-Vinculin (mouse monoclonal, Sigma, V9131, 1:10000).

Esophageal tissues from 52 malignant tumors and 28 benign or normal subjects were purchased from US Biomax, Inc. (no. ES809; Rockville, MD, USA). Colon tissues from University Hospital and US Biomax, Inc. (no. BC05002) were also used for the analysis. Avidin-biotin immunohistochemical analysis was performed as previously described.^{46 (link)} Briefly, serial sections of human colon disease tissue slides and esophageal tissues were subjected to antigen retrieval and probed with anti-LKB1 (Abcam, 1 : 50), anti-PLK1, or anti-PLK1 (T210) antibody (Abcam, 1 : 100) at 4 °C overnight. Biotin-conjugated anti-mouse or -rabbit IgG (Jackson Laboratories, Bar Harbor, ME, USA) was applied in a 1 : 300 dilution for 1 h at room temperature. Bound secondary antibody was further incubated with avidin-HRP (Vector Laboratories, Burlingame, CA, USA). After 3,3′-diaminobenzidine (DAB) staining, slides were counterstained with hematoxylin. Staining regions were reviewed by three different investigators and categorized as negative, weak, moderate, or strong. For determining the H-score, PLK1 (T210)-stained tissues were scored by calculating the product of the percentage of cells staining at each intensity level and the intensity level (0, negative; 1+, weak; 2+, moderate; and 3+, strong). An H-score was then calculated by summing the individual intensity level scores.^{49 (link)}