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. 2002 Feb 1;22(3):815–824. doi: 10.1523/JNEUROSCI.22-03-00815.2002

Fig. 5.

Fig. 5.

Constitutively hypophosphorylated pRb inhibits the S-phase entry, but not the survival, caused by N-myc.A, Constitutively hypophosphorylated pRb rescues the N-myc-induced BrdU incorporation. Neurons were coinfected with 50 MOI N-myc adenovirus and 50–200 MOI pRb adenovirus, incubated with BrdU 1 d later, and then analyzed immunocytochemically for BrdU and neurofilament-M after an additional 2 d. As a control, neurons were coinfected with 200 MOI of a β-galactosidase adenovirus (LacZ). Quantitation was performed as described in Figure 2D. *p < 0.05; Student'st test, comparing N-myc plus pRb versus N-myc plus β-galactosidase. B, pRb has no effect on N-myc-induced survival after NGF withdrawal. Neurons were infected with 200 MOI N-myc in the presence or absence of 100 or 200 MOI hypophosphorylated pRb adenovirus, and 2 d later NGF was withdrawn. Neurons were stained with Hoechst, and intact, nonapoptotic nuclei were quantitated by counting at least 300 cells in each of three random fields.C, AdN-myc decreases p75NTR expression and inhibits the activation of JNK. Immunoblots of sympathetic neurons infected with 100 or 400 MOI of N-myc adenovirus in the presence (right panel) or absence (left panel) of NGF. Left panel, Neurons were infected with N-myc adenovirus for 48 hr and then withdrawn from NGF for an additional 24 hr. Equal amounts of protein were analyzed for expression of the p75NTR (α-p75), the activated, phosphorylated form of JNK (α-P-JNK), or for total MAP kinase protein levels (α-MAPK). Note that although p75NTR and phospho-JNK levels were reduced on infection with N-myc, total MAP kinase levels remained the same. Right panel, Neurons were infected with N-myc adenovirus and maintained in NGF for 72 hr before Western blot analysis. Immunoblots were first probed with an antibody to p75NTR and then reprobed with an antibody for total α-tubulin to demonstrate that similar amounts of protein were present in each lane. Note the concentration-dependent decrease in p75NTR levels in neurons infected with the N-myc adenovirus. Similar results were obtained in four independent experiments.