Figure 2.
Immunocytochemical and Western blot analysis of SMN protein in dissociated cultures of mouse embryonic spinal cord. (A) Phase contrast micrograph of cultured cells after 7 days in vitro (Bar = 30 μm). (B) abSMN immunoreactivity is limited to neuronal cell bodies. (C) Concurrent labeling of cell shown in B with mouse anti-Islet 1 antibody indicates that SMN is present within spinal motor neurons. (D) Immunoblot of cell extracts prepared from spinal cord cultures. Samples (8 μg total protein) were probed with 125 ng/ml abSMN. Like SY5Y cells, mixed neuron/glia cultures from spinal cord demonstrated a 34-kDa band in the insoluble protein fraction. Lane 1: SY5Y cell extract, insoluble nuclear fraction. Lane 2: Ventral spinal cord culture, insoluble fraction. Lane 3: Dorsal spinal cord culture, insoluble fraction. Lane 4: Cortical glia culture, insoluble fraction. Lane 5: Ventral spinal cord culture, soluble fraction. Lane 6: Dorsal spinal cord culture, soluble fraction. Lane 7: Cortical glia culture, soluble fraction.