Characterization of motor neuron differentiation. HBG3 cells were induced to differentiate following the procedure as in 1A. Lysates from undifferentiated and differentiated cells were resolved on an SDS-PAGE and transferred to a membrane. The blot was probed with antibodies against pan-neuronal (neurofilament, NF) and motor neuron markers (Hb9 and ChAT). The same blot was stripped and re-probed with anti-ERK antibodies for protein loading control (A). Differentiated ES cells were dissociated and processed for indirect immunofluorescence staining for neurofilament (NF, in red), ChAT (in red), Islet-1 (in red), and DAPI (for nuclei in blue). Scale bar = 50 μm. U = undifferentiated cells and D = differentiated cells.