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Figure 1


Fig. 1. Characterisation of anti-Cep68 and anti-Cep215 antibodies. (A) Primary structure of human Cep68L/S. Note that Cep68L comprises a 137 aa insertion compared with Cep68S (at residue 492) and both isoforms are predicted to form a C-terminal globular domain (at aa 618-757 in Cep68L and at aa 454-620 in Cep68S; grey). The horizontal line (antigen) denotes the region used for antibody production. The sequence data for Cep68 are available from Ensembl [accession no. ENST00000377990 (Cep68L) and ENST00000260569 (Cep68S)]. (B) Primary structure of human Cep215. Two isoforms with 1893 and 1814 residues, respectively, have been described; they are identical between residues 1 and 1396, followed by non-identical stretches of 259 (isoform 1) and 180 residues (isoform 2), and identical C-termini (starting at residue 1655 in isoform 1 and 1576 in isoform 2). Both isoforms are predicted to contain several coiled-coil regions (black, CC) and a microtubule association region (grey). The horizontal line (antigen) denotes the region used for antibody production. The sequence data for Cep215 cDNAs are available from NCBI [accession no. NM_018249 (isoform 1) and NM_001011649 (isoform 2)]. (C) Antibodies directed against Cep68 (R170) and corresponding pre-immune serum were tested on western blots of total cell lysates of HeLaS3, U2OS and 293T cells (left) and centrosome preparations from KE37 cells (centre). In the latter case, proteins show a retarded electrophoretic mobility because of the high sucrose concentration present in centrosome preparations. Arrowheads indicate Cep68L and -S. Right, western blot showing efficient depletion of Cep68 from U2OS cells by 48 hours siRNA, using oligonucleotide duplexes 262 or 263. (D) Antibodies directed against Cep215 (R174) and corresponding pre-immune serum were tested on western blots of total cell lysates of HeLaS3, U2OS and 293T cells (left) and centrosome preparations from KE37 cells (centre). In the latter case, proteins show a retarded electrophoretic mobility because of the high sucrose concentration present in centrosome preparations. Arrowheads indicate Cep215. Right, western blot showing efficient depletion of Cep215 from U2OS cells by 72 hour siRNA, using oligonucleotide duplexes 282 or 283. GL2 treatment is used for control and blotting for {alpha}-tubulin illustrates equal loading.