Biochemical and Biophysical Research Communications
Alternative splicing generates a novel non-secretable cytosolic isoform of NELL2
Section snippets
Materials and methods
Antibodies. Monoclonal antibodies specific for EGFP (B-2) and a rabbit anti-PKCβI antibody (C-16) were obtained from Santa Cruz Biotechnology. Rabbit polyclonal anti-actin antibody was purchased from Sigma.
RNA extraction and RT-PCR. Total RNA was isolated from tissues of adult male Sprague–Dawley rats (SD, 14 weeks old) using TRIZOL (Invitrogen) according to the manufacturer’s protocol. One microgram of total RNA was reverse-transcribed using SuperScript II RT (Invitrogen), random hexamers (50
A novel alternative splicing variant of the rat NELL2 gene
During RT-PCR analysis of NELL2 expression in the rat thymus, two different NELL2 transcripts were detected: (1) the expected NELL2 mRNA (468 bp), which corresponds to the known secreted NELL2 mRNA [4], [9], [10], and (2) a shorter 339-bp PCR fragment (Fig. 1A). Since PCRs were carried out with specific primers corresponding to exons 2 and 5 of rat NELL2 cDNA, alternative splicing was proposed to account for the presence of this fragment. Fig. 1A shows the genomic structure of the rat NELL2 gene
Discussion
Here we report a novel cytosolic form of rat NELL2 (cNELL2) that is generated by alternative splicing. In several previous studies in which human and rat tissues [2], [3], [4], [11] were analyzed by either Western or Northern blotting, only the secreted form of NELL2 was detected. Two technical problems have made studies of cNELL2 difficult: First, the expression level of cNELL2 is much lower than that of secreted NELL2, as demonstrated by our RT-PCR results which showed differences in the
Acknowledgments
This work was supported by a grant from the Basic Research Program of the Korean Science & Engineering Foundation (R13-2005-012-01003-0), and the Korean Research Foundation (KRF-2002-CS0045 and KRF-2006-005-J04204). The nucleotide sequences reported in this paper have been submitted to the GenBank™ databases under Accession No. EF110908.
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These authors contributed equally to this work.