Fig. 3. Western-blot analysis of the subcellular distribution of PtNSF using affinity-purified Abs against yeast and Paramecium NSF. Aliquots of 50 µg of whole cell homogenates (lanes 1,5), 100,000 g supernatant (lanes 2,6), 100,000 g pellet (lanes 3,7), isolated cortices (lanes 4,8), and of 100 ng of recombinant Sec18p (lane 9) were separated on a 10% SDS polyacrylamide gel and electroblotted onto nitrocellulose membranes. Immunoreactions were carried out with either affinity-purified and crossreactive polyclonal Abs against Sec18p (lanes 1-4) (Mayer et al., 1996) or with affinity-purified peptide-specific Abs against PtNSF1 (lanes 5-9), as described in the Materials and Methods. Note that each of the two Abs immunoreact with a protein band at 84 kDa (arrow) not only in whole cell homogenates but also in particulate fractions rich of ER. In addition, the polyclonal Ab against SEC18p (anti-yeast NSF) also recognizes some bands at 30 kDa (lanes 1-4), which might be the result of proteolytic activity or instability of the NSF protein itself. By using the peptide-specific Ab against Paramecium PtNSF, some higher molecular weight bands are also reactive, which may represent heteromeric complexes of NSF, which still have to be analysed in more detail.

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