Psgl 1

The total protein concentration was measured using a bicinchoninic acid assay kit (Beyotime Institute of Biotechnology, Haimen, China). In brief, 30 μg of proteins were loaded into each lane of sodium dodecyl sulfate polyacrylamide gels, separated by electrophoresis, and then transferred to polyvinylidene fluoride membranes (Pall Corporation, Beijing, China), which were incubated with primary Abs against IRAK4, phospho-IRAK4, P38, phospho-P38, JNK, phospho-JNK, TRIF, IRF3, phospho-IRF3, NF-κB p65, phospho-NF-κB p65, IKKα, IKKβ, phospho-IKKα/β, IkBα, and phospho-IkBα (Cell Signaling Technology, Inc., Beverly, MA, USA; all diluted to 1:1000), as well as GAPDH (1:5000) and PSGL-1 (Santa Cruz Biotechnology, Inc.; 1:1000). Anti-rabbit or mouse horseradish peroxidase-linked Ab (ZSGB-BIO Technology Co., Ltd., Beijing, China; 1:8000) was used as the secondary Ab. Detection was performed using a chemiluminescence kit (Advansta, Inc., Menlo Park, CA, USA). Densitometry of proteins was analyzed with Gel-Pro software (Media Cybernetics).

In order to evaluate binding sites and their protein backbones cells on the cell surface of OH-1 cells, cultured OH-1-LUC/mCherry cells were detached with Cell Dissociation Buffer (Gibco, Carlsbad, US) and stained for sialyl Lewis A (abcam, dilution 1∶1000), CEA (Cell Signalling, dilution 1∶200), CD44 (AbD Serotec, dilution 1∶1000), PSGL-1 (Santa Cruz, dilution 1∶200), E- and P-selectin binding sites themselves using E- and P-selectin fusion proteins (R&D Systems, dilution for E-selectin 1∶1000, for P-selectin 1∶100), EpCAM (Dako, dilution 1∶59), Muc18 (GeneTex, 1∶1800) and NCAM (R&D Systems, dilution 1∶1000). The corresponding isotype controls (IgG1 for PSGL-1, EpCAM, Muc18, CEA, E- and P-selectin; IgG2a for CD44 and NCAM; IgM for sialyl Lewis A) were incubated in parallel. The antibody expression was determined with FACS CALIBUR flow cytometer (Becton Dickinson, Heidelberg, Germany) and analyzed with Win MDI 2.9 software.
In vivo grown OH-1 cells from a primary OH-1-Luc/mCherry tumor were investigated by FACS analysis. Cells were double labelled for anti-CEA and a human E- or P-selectin fusion protein.