Mouse Anti-CD164 Recombinant Antibody (clone 67D2) (CAT#: HPAB-0077-YJ)

Figure 1 Expression of CD164 epitopes by stromal reticular cells and MS.5 transfectants.

Immunofluorescence staining of cultured human bone marrow stromal reticular cells with CD164 mAbs: mAb 103B2/9E10 (a), mAb 105A5 (c), mAb N6B6 (e), and mAb 67D2 (g). Isotype-matched negative control mAbs for each CD164 mAb are shown as insets (b, d, f, and h).

Doyonnas, R., Chan, J. Y. H., Butler, L. H., Rappold, I., Lee-Prudhoe, J. E., Zannettino, A. C.,... & Watt, S. M. (2000). CD164 monoclonal antibodies that block hemopoietic progenitor cell adhesion and proliferation interact with the first mucin domain of the CD164 receptor. The Journal of Immunology, 165(2), 840-851.

Figure 2 Immunoblot analyses of CD164.

Non-transfected MS.5 cells (lane 1) or MS.5 cells transfected with the different CD164 splice variants are shown: CD164(E1-6) (lane 2), CD164(ED4) (lane 3), and CD164(ED5) (lane 4) were lysed in Laemmli SDS lysis buffer containing 5 mM DTT and immunoblotted with N6B6 (lanes 1-4). Human bone marrow cells (lanes 5 and 8), CD341 purified cord blood cells (lanes 6 and 9), or cultured bone marrow stromal reticular cells (lanes 7 and 10) were lysed and immunoblotted with N6B6 (lanes 5-7) or 67D2 (lanes 8-10). Molecular mass markers were myosin (220 kDa), phosphorylase b (97.4 kDa), BSA (69 kDa), OVA (46 kDa), and carbonic anhydrase (31 kDa).

Doyonnas, R., Chan, J. Y. H., Butler, L. H., Rappold, I., Lee-Prudhoe, J. E., Zannettino, A. C.,... & Watt, S. M. (2000). CD164 monoclonal antibodies that block hemopoietic progenitor cell adhesion and proliferation interact with the first mucin domain of the CD164 receptor. The Journal of Immunology, 165(2), 840-851.

Figure 3 Immunoblot analyses of CD164 from hemopoietic cell lines.

A, TF1 (lane 1), KG1a (lane 2), KG1b (lane 3), U937 (lane 4), THP1 (lane 5), CEM (lane 6), and RPMI 1788 (lane 7) cells, were lysed in SDS-Laemmli lysis buffer containing 5 mM DTT and resolved by 10% SDS-PAGE before immunoblotting with N6B6, 103B2/9E10, or 105A5 as indicated below each blot. B, KG1a cells were lysed with 1% SDS (lane 1) or 1% Triton X-100 (lanes 2 and 3). The Triton X-100-insoluble fraction was solubilized in SDS before analysis. The distribution of the CD164 monomer and dimer and the.220-kDa complex between the Triton X-100-soluble (lane 3) and Triton X-100-insoluble (lane 2) fractions was assessed by immunoblotting with the four CD164 mAbs as indicated below each blot.

Doyonnas, R., Chan, J. Y. H., Butler, L. H., Rappold, I., Lee-Prudhoe, J. E., Zannettino, A. C.,... & Watt, S. M. (2000). CD164 monoclonal antibodies that block hemopoietic progenitor cell adhesion and proliferation interact with the first mucin domain of the CD164 receptor. The Journal of Immunology, 165(2), 840-851.

Figure 4 Sialidase and O-sialoglycoprotease treatment of KG1a cells identifies three classes of CD164 epitopes.

KG1a cells were untreated or enzymatically digested for 1 h at 37°C, then stained with CD164 mAbs (unbroken lines) or isotype-matched negative control mAbs (dotted lines). CD34 mAbs class I (My10), class II (QBEND-10), and class III (Tu¨k3) were used in parallel to stain KG1a cells, and values are shown in the table with their isotype-matched negative controls. Cells were analyzed for fluorescence staining using a FACScalibur flow cytometer. For each Ab, the upper histogram represents the staining of untreated cells; the middle histogram shows the staining of sialidase-treated cells; and the lower histogram shows the staining of O-sialoglycoprotease-treated cells. Values represent the median fluorescence intensity (MFI) 6 SD for three individual experiments.

Doyonnas, R., Chan, J. Y. H., Butler, L. H., Rappold, I., Lee-Prudhoe, J. E., Zannettino, A. C.,... & Watt, S. M. (2000). CD164 monoclonal antibodies that block hemopoietic progenitor cell adhesion and proliferation interact with the first mucin domain of the CD164 receptor. The Journal of Immunology, 165(2), 840-851.