Fig. 1. Characterization of exogenous NF subunits and their distribution in DRG neurons. (A) Coomassie Blue staining (CBB) following SDS-gel electrophoresis of NFs isolated from bovine spinal cords, and immunoblot analysis of this preparation (NFs) following biotinylation and of chromatographically separated NF-L as indicated. (B) Representative DRG neuron 2 hours after microinjection with a mixture of biotin-L and fluorescein-conjugated tracer, followed by extraction with Triton X-100 and immunostaining with an anti-biotin antibody followed by a Texas-Red-conjugated secondary antibody. Notice the retention of biotin-L along axons (arrows), indicating its incorporation into Triton-X-100-insoluble structures. (C) A second microinjected neuron processed for anti-biotin immunoreactivity following extraction under conditions that induce splaying of NFs, more clearly revealing individual filamentous profiles. (D) Region of a DRG axon extracted with Triton X-100 and processed for immuno-EM (directed against biotin) 2 hours after injection. Notice the association of colloidal gold particles with filamentous profiles (arrows). (E) Representative DRG neurons (arrows), interspersed with non-neuronal cells, fixed and processed for SMI-31 immunoreactivity 3 days after plating. One neuron (large arrowhead) was microinjected 2 hours before fixation with biotin-L. Neurons in culture have expressed and transported endogenous NFs by this time, as shown by perikaryal and axonal SMI-31 immunoreactivity. The single microinjected neuron also displays prominent perikaryal and axonal biotin immunoreactivity. (F) Immunoblot probed with an antibody (SMI-32) against non-phosphorylated epitopes common to NF-H and NF-M, of material immunoprecipitated from a homogenate of a NB2a/d1 culture that had been transfected the previous day with GFP-M. Notice the appearance of an SMI-32-immunoreactive band of 172 kDa (arrow), which corresponds to the expected migratory position of NF-M (145 kDa) conjugated to GFP (30 kDa) (Yabe et al., 2001a,b). (G) Perikaryon and proximal axonal neurite of a cell transfected with GFP-M the previous day. Notice the incorporation of GFP-M into filamentous profiles.