Analysis of developed anti-EB1 antibodies compared to the known anti-tubulin antibodies.

Abstract

<p>(A) Western-blot analysis of the indicated proteins (15 ng) separated on 10% SDS-PAGE using a commercial anti-EB1 antibody (anti-total EB1, raised against amino-acids 107–268 of mouse EB1), the presently developed antibodies (anti-Tyr EB1 and anti-deTyr EB1), and tubulin antibodies. Detyrosinated EB1 was obtained from recombinant EB1 using carboxypeptidase A. Tyrosinated and detyrosinated tubulin were obtained from purified brain tubulin, using respectively TTL and carboxypeptidase A. Both anti-Tyr EB1 and anti-deTyr EB1 are highly specific. (B) Double immunostaining with anti-total EB1 antibody and anti-Tyr EB1 on fibroblasts after transfection of plasmids allowing expression of either tyrosinated or detyrosinated EB1 with EGFP at the N-terminus. The transfected cells were detected by EGFP signal. Anti-Tyr EB1 is highly specific of tyrosinated form of EB1. (C) Immunostaining of endogenous EB1 in astrocytes with anti-Tyr EB1 and anti-total EB1.</p