Anti cenpf

Cells or tumors were lysed in ice-cold lysis buffer containing 100 mM Tris-HCl (pH 6.8), 4% sodium dodecyl sulfate (SDS), 20% glycerol, 2% hydroxyethanal, and clarified by centrifugation at 15000 rpm for 15 min. Equal amounts of cell lysate protein (20 μg of protein per lane) were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinyl difluoride (PVDF) membranes (0.45 μm; Millipore, Bedford, MA). For immunoblot analysis, the membranes were probed with specific primary antibodies, anti-CENPF (Abcam, Cambridge, MA), anti-cyclinD1 (Abcam, Cambridge, MA), anti-c-Myc (Abcam, Cambridge, MA), anti-CDK2 (Cell Signaling Technology, Beverly, MA), anti-CDK4 (Cell Signaling Technology, Beverly, MA), anti-CDK6 (Cell Signaling Technology, Beverly, MA), and anti-β-actin antibody (Cell Signaling Technology, Beverly, MA), and secondary antibodies conjugated to HRP (ZhongShan JinQiao, Beijing, China). Immunocomplexes on the membrane were visualized using Image Quant LAS 4000 (GE Healthcare, Tokyo, Japan) with Immobilon® Western HRP Substrate luminol reagent and peroxide solution (Millipore).

Tissue microarray chips containing 99 samples of pancreatic cancer and 71 samples of paired normal pancreatic tissue were purchased from Outdo Biotech (Shanghai, China). The characteristics of the patients are shown in Supplementary File S1. Immunohistochemistry (IHC) staining was performed as previously described [23 (link)]. Microarray chips were stained with anti-CENPF (Abcam), anti-SCEL, anti- GLUT-1, anti-SLC6A14, anti-TMC7 (Thermofisher, Waltham, MA USA), anti-TMPRSS4 and anti- MASpin (Gene Tex, Irvine, CA, USA) antibodies. Control staining with only secondary antibodies was performed to ensure specificity. Rabbit IgG polyclonal-Isotype control (Abcam) was used as the negative control. The score for staining was independently assessed by two experienced pathologists based on the integrated staining intensity and the proportion of positive cells. The final score was determined by adding the staining intensity score and the average proportion of positive cells score; the final score ranged from 0 to 7. For the purpose of further analysis, the samples with a score of 0–3 were defined as low expression, while the samples with scores of 4–7 were defined as high expression.