Mouse Anti-CD200R1 Recombinant Antibody (clone OX102) (CAT#: FAMAB-1617CQ)

Figure 1 Generation of mAb OX102 and Biochemical Analysis of the OX2R

(A) Staining of rat RPCs with mAbs OX102(bold) and an isotype-matched negative control (OX21). (B) Rat RPCs were preincubated with OX45(anti-rat CD48) or OX102 mAbs and tested for their ability to bind rOX2CD4d314-coated beads. rOX2 binding was blocked back to a negative control (CD4d314-coated beads, thin line) by OX102 (bold line) but not by the control OX45 (dotted line). (C) The rOX2R was purified from deoxycholate solubilized crude rat spleen Tween-40membrane preparations by immunoprecipitation and further resolved under nonreducing (NR) and reducing (R) conditions by SDS-PAGE.(D) Purified rOX2R was left untreated (U) or was incubated with PNGaseF for the indicated times, then resolved under nonreducing conditions by SDS-PAGE, blotted onto nitrocellulose, and probed with OX102 mAb.

Wright, G. J., Puklavec, M. J., Willis, A. C., Hoek, R. M., Sedgwick, J. D., Brown, M. H., & Barclay, A. N. (2000). Lymphoid/neuronal cell surface OX2 glycoprotein recognizes a novel receptor on macrophages implicated in the control of their function. Immunity, 13(2), 233-242.

Figure 2 The OX2R Binds OX2.

(A) HEK 293T cells were transfected with rat OX2R cDNA and stained for OX102 mAb (bold line), the endogenous human MHC class I with mAb W6/32 (dotted line), and a negative control mAb (OX21, thin line). Mock transfected cells stained with OX102 mAb showed no staining greater than the negative control(data not shown).(B) rOX2R cDNA transfected 293T cells preincubated with OX102 (bold), but not W6/32(dotted), were able to block binding of rOX2-coated beads back to a negative control (thin line). Mock transfected cells did not bind rOX2-coated beads (data not shown).(C) Staining of rat thymocytes with mAbs OX1(dotted line), OX2 (bold line), and an isotype-matched negative control OX21.(D) Rat thymocytes preincubated with mAbOX2 (bold), but not OX1 (dotted), were able to block the binding of rat OX2R-coated beads back to the negative control (thin line).The negative controls in (B) and (D) are CD4d3+4-coated beads.

Wright, G. J., Puklavec, M. J., Willis, A. C., Hoek, R. M., Sedgwick, J. D., Brown, M. H., & Barclay, A. N. (2000). Lymphoid/neuronal cell surface OX2 glycoprotein recognizes a novel receptor on macrophages implicated in the control of their function. Immunity, 13(2), 233-242.

Figure 3 Mouse Anti-CD200R1 Recombinant Antibody (clone OX102) in WB

Anti-phosphotyrosine (a-PY) Western blot of OX102 mAb immunoprecipitates from rat RPCs that were left unstimulated (2) or were stimulated for 10 min with 1 mM sodium pervanadate (1). The bands at 50 and 25 kDa, in both lanes, are the OX102 mAb heavy and light chain (respectively), which were visualized due to the peroxidase-conjugated anti-mouse secondary antibody.

Wright, G. J., Puklavec, M. J., Willis, A. C., Hoek, R. M., Sedgwick, J. D., Brown, M. H., & Barclay, A. N. (2000). Lymphoid/neuronal cell surface OX2 glycoprotein recognizes a novel receptor on macrophages implicated in the control of their function. Immunity, 13(2), 233-242.

Figure 4 OX2R Is Restricted to the Myeloid Lineage in Rat and Can Be Tyrosine Phosphorylated upon Pervanadate Treatment.

(A) Rat dendritic cells were defined as highly granular, OX62 mAb-positive cells in thoracic duct lymph collected from mesenteric lymphadenectomized rats (Pugh et al., 1983) and were stained with OX102 (bold line) and an isotype-matched negative control (OX21).(B) Adult CNS leukocyte and microglial cell characterization. Gates are as follows: R1, microglia (CD45lo, CD11b/c+); R2, CNS-associated macrophages/monocytes/granulocytes (CD45hi, CD11b/c+); R3, lymphocytes (CD45hi, CD11b/c-).(C) R1-gated microglia are stained with biotinylated OX102 (bold line) or an isotype-matched negative control. Around 20% of cells in R2 were OX2R+, representing CNS vascular associated macrophages and blood granulocytes, and cells in R3, mostly lymphocytes, were not stained(data not shown).

Wright, G. J., Puklavec, M. J., Willis, A. C., Hoek, R. M., Sedgwick, J. D., Brown, M. H., & Barclay, A. N. (2000). Lymphoid/neuronal cell surface OX2 glycoprotein recognizes a novel receptor on macrophages implicated in the control of their function. Immunity, 13(2), 233-242.