Study of endogenous EB1 C-termini in fibroblasts and brain from wild type and TTL-deficient mouse.

<p>Western-blot analysis of the indicated control proteins (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033490#pone-0033490-g001" target="_blank">figure 1</a>) or extracts. (A) Immunoprecipitation of endogenous EB1 from wild type (TTL^+/+) or TTL-deficient MEFs using anti-total EB1 antibody, and analysis with anti-Tyr EB1 (1∶15000), anti-detyr EB1 (1∶200), and anti-total EB1 (1∶2000). EX: crude extract; SN: supernatant after immunoprecipitation; IP: immunoprecipitated fraction. No detyrosinated EB1 could be detected in the IP fractions. Note that anti-total EB1 antibody being less sensitive than anti-Tyr EB1, EB1 failed to be detected in crude extract (upper panel). (B) Immunodepletion of tyrosinated EB1 with anti-Tyr EB1 (IP 1 to 4) in brain extracts from wild type and TTL-knockout mice, followed by immunoprecipitation of the remaining EB1 with anti-total EB1 (IP5), and analysis with anti-total EB1 (1∶2000). No remaining EB1 could be detected after tyrosinated-EB1 immunodepletion. (C) Tyrosinated and detyrosinated tubulin pools in brain extracts from wild type and TTL-deficient mice were analyzed using anti-α tubulin (1∶10,000), anti-tyrosinated tubulin (YL_1/2, 1∶20,000), and anti-detyrosinated tubulin (L₄, 1∶20,000).</p

Analysis of C-termini of recombinant EB1 forms overexpressed in wild type and TTL-deficient fibroblasts.

<p>Western-blot analysis of the indicated control proteins (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033490#pone-0033490-g001" target="_blank">figure 1</a>) and of fractions of immunoprecipitation experiments carried out on cells transfected with cDNA encoding different EB1 forms. EX: crude extract; SN: supernatant after immunoprecipitation; IP: immunoprecipitated fraction. (A) transfection of fibroblasts (NIH3T3) with plasmids encoding tyrosinated EB1 fused with GFP at the N-terminus (GFP-EB1), followed by immunoprecipitation using anti-GFP antibody and analysis using anti-Tyr EB1 (1∶15000) and anti-deTyr EB1 (1∶200). No detyrosinated GFP-EB1 could be detected. (B) Transfection of fibroblasts with cDNA encoding GFP-EB1 ending with the C-terminus of α-tubulin GEEEGEEY (GFP-EB1-CterTub), followed by immunoprecipitation with anti-GFP antibody and analysis using anti-Tyr Tub (1∶20,000) and anti-deTyr Tub (1∶20,000). NIH3T3 were used as TTL^+/+ cells and MEFs isolated from TTL null mice were used as TTL^−/−. A very low quantity of detyrosinated protein ending with α-tubulin residues was detected (upper band in IP fractions of lower panel).</p

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

<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