Characterization of antibodies raised against Nogo receptor family members. A, Domain alignment of the Nogo receptor family members NgR1 (473 amino acid residues), NgR2 (420 amino acid residues), and NgR3 (445 amino acid residues). Nogo receptors are composed of eight canonical leucine-rich repeats (LRR1-LRR8) flanked by cysteine-rich LRRNT and LRRCT subdomains. The highly conserved LRRNT+LRR+LRRCT domains are connected via a more variable unique domain (stalk) to a GPI anchor for membrane attachment. We raised polyclonal antibodies against the distal part of the LRRCT and the unique domain of each of the three NgR family members. B, Immunocytochemistry (ICC) under nonpermeabilizing conditions of myc-tagged NgR1, NgR2, and NgR3 in transfected COS-7 cells reveals abundant cell surface localization of NgR1 (B1), NgR2 (B2), and NgR3 (B3). Anti-NgR1 (B4), anti-NgR2 (B8), and anti-NgR3 (B12) immune sera strongly and selectively react with their cognate antigens. No cross-reactivity with other NgR family members is observed. C, Western blotting with anti-NgR1, anti-NgR2, and anti-NgR3 immune sera allows for selective detection of recombinant (COS-7) and endogenously (adult rat brain) expressed receptors. All three NgR family members are expressed abundantly in the adult brain. Of note, endogenously expressed NgR1 and NgR2 are detected as single bands and migrate at an apparent molecular weight of 65 kDa. Recombinant NgR1 and NgR2 occur as multiple variants between 55 and 70 kDa (Walmsley et al., 2004). Multiple variants are found for recombinant and endogenously expressed NgR3. Scale bar, 20 μm.