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. 1998 May 26;95(11):6492–6497. doi: 10.1073/pnas.95.11.6492

Figure 1.

Figure 1

Characterization of abSMN. (A) Immunoblots of E. coli expressed SMN fusion proteins and subcellular fractions of human neuroblastoma cells probed with abSMN (lanes 1–8) or chicken anti-human SMN antibody C3 (lanes 9–10). AbSMN reacts with two different fusion constructs expressing wild-type SMN (fusion proteins 1 and 2) but not with extracts from bacteria lacking the SMN cDNA insert or extracts from cells obtained before induction of protein expression. Subcellular fractions of SY5Y cells were probed by using the same abSMN antibody concentration. Lane 1: BL21DE3 bacteria, negative control (2 μg). Lane 2: BL21DE3 bacteria expressing fusion protein 1 (2 μg). Lane 3: DH5α bacteria, negative control (5 μg). Lane 4: DH5α bacteria expressing fusion protein 2 (5 μg). Lane 5: SY5Y cells, total cell lysate (20 μg). Lane 6: SY5Y cells, cytosolic fraction (20 μg). Lane 7: SY5Y cells, soluble nuclear extract (20 μg). Lane 8: SY5Y cells, insoluble nuclear material (20 μg). Lane 9: SY5Y cells, cytosolic fraction (20 μg). Lane 10: SY5Y cells, insoluble nuclear material (20 μg). (B) Immunocytochemical localization of SMN in human SY5Y neuroblastoma cells determined by using double-label indirect immunofluorescence. (i) Fixed cells viewed when using Hoffman modulation (Bar = 20 μm). (ii) abSMN (200 ng/ml) immunostaining demonstrates moderate cytoplasmic labeling and strong labeling of nuclear gems. (iii) Simultaneous immunostaining with monoclonal anti-p80-coilin antibody (1:150) shows that abSMN reactive gems are often closely associated with nuclear coiled bodies. (C) Preadsorption studies of abSMN. Detection of SMN protein by immunoblot analysis persists after preincubation of abSMN with an exon 4 oligopeptide (lanes 1–4), whereas preadsorption with the appropriate exon 2 peptide eliminates SMN immunoreactivity (lanes 5–8). Lanes 1 and 5: BL21DE3 bacteria, negative control (2 μg). Lanes 2 and 6: BL21DE3 bacteria, fusion protein 1 (2 μg). Lanes 3 and 7: SY5Y cells, cytosolic fraction (20 μg). Lanes 4 and 8: SY5Y cells, insoluble nuclear material (10 μg).