Fig. 2.

Expression profile of neuronal markers and cell cycle regulators in serum-deprived PC12-Nex1 cells. (a) Phase-contrast micrographs of serum-deprived PC12-Nex1 cells reveal a maintained neuronal phenotype accompanied by a flattened and wide cell body and significant neurite outgrowth. Serum-grown PC12-Nex1 cells, τ = 0 days; serum-deprived PC12-Nex1 cells, τ = 21 days. (b) Nex1 expression and that of specific neuronal markers remain constant during serum deprivation of PC12-Nex1 cells. PC12-Nex1 cells extracts isolated at successive times after serum deprivation as indicated above the panel were subjected to immunoblot analyses using appropriate antibodies described in Table 1 and corresponding secondary antibodies. The antigen—antibody complexes were detected by chemiluminescence. The membrane was stripped with Restore™ western blot stripping buffer and re-probed with the anti-βIII tubulin, anti-α tubulin, and anti-GAP-43 antibodies separately. (c) Constitutive expression of Nex1 results in sustained expression of specific CDK inhibitors upon serum deprivation. Immunoblot analyses using PC12-Nex1 cell extracts were performed as described above with antibodies described in Table 1. The membrane was stripped with Restore™ stripping buffer and re-probed with the anti-p27^cip1, anti-p16^INK4a and CDC47 antibodies separately. Data shown are representative of at least three independent experiments.