Figure 2.

Immunoblot analysis of mouse and human CLEC3A. A–G, recombinant mouse and human full-length CLEC3A (CLEC3A-flc), CLEC3A-Ex23, CLEC3A-Ex3, chondroitinase ABC–treated recombinant full-length CLEC3A, tissue extracts from a mixture of forelegs and ribs of a newborn mouse (newborn) and from the tail of a 2-year-old mouse (adult), and human cartilage extracts were separated by SDS-PAGE under reducing (+SH) and nonreducing (−SH) conditions and visualized by Coomassie staining or by immunoblot with Affinity-purified antibodies against murine CLEC3A-Ex3 (A–D) or human full-length CLEC3A (flc) (E–G). Both recombinant and tissue CLEC3A occur mainly as monomer. Faint bands with a molecular mass around 40 kDa most likely represent dimeric CLEC3A. Note that recombinant CLEC3A possesses a 3.5-kDa strepII-tag. C, incubation of CLEC3A with chondroitinase ABC (+) led to the loss of the higher diffuse band, indicating that CLEC3A carries chondroitin sulfate and/or dermatan sulfate side chains. Additional bands in the Coomassie Blue–stained gel of chondroitinase ABC–treated CLEC3A originate from the enzyme preparation and from albumin which was used as a carrier (all samples +SH). E, human cartilage extracts were separated by SDS-PAGE followed by immunoblot with Affinity-purified antibodies against human CLEC3A. 1–4, extraction buffer 1–4 (see “Experimental procedures”). Please note that the human antibodies react only weakly with nonreduced monomeric CLEC3A.