Figure 1

Sema5B antibody specificity. (a) Schematic illustration of Sema5B including the region in the N-terminus used to generate the antibody. The amino acids bordering the sema domain and thrombospondin repeats are indicated. (b) Lysates from human embryonic kidney (HEK)293 cells transfected with hemaglutinin (HA)-Sema5B were separated by SDS-PAGE and immunoblots probed with antibodies specific to Sema5B or HA. Antibodies recognize a predominant band at approximately 150 kDa corresponding to HA-Sema5B. (c) Anti-5B does not recognize a full-length mouse Sema5A extracellular domain Fc-human fusion protein (Sema5A-Fc). Purified Sema5A-Fc and Fc proteins (kindly provided by Dr D Sretavan) were separated by SDS-PAGE and immunoblots probed with antibodies specific to Sema5B (anti-5B) or anti-human Fc chain (anti-Fc). Some crossreactivity was observed in the Fc lane, most likely between the anti-rabbit IgG secondary and the large amount of human Fc loaded on the gel. Sema5A-Fc ran at the expected molecular weight of ~170 Kda. (d) Sema5B is proteolytically processed in vivo. Lysates from P1 mouse brains and hippocampal neurons cultured for 0 and 21 days in vitro (DIV) were separated by SDS-PAGE and immunoblots probed with anti-5B. Numerous repeatable bands were observed indicating proteolytic processing of Sema5B in the nervous system. N = 4 brains.