Figure 6. Numb Interacts With aPKC to Promote GCP Chemotaxis.

(A) PKCζ interacts with Numb. 293T cells were transfected with pcDNA3-myc-Numb plasmids, and lysates were precipitated with control IgG or anti-Myc then blotted with anti-PKCζ. Membrane was reprobed with anti-Numb.

(B) PKCζ interacts with Numb in cerebellar GCPs. GCP lysates (P7) were immunoprecipitated with anti-PKCζ or control IgG then blotted with anti-Numb. Membrane was reprobed with anti-PKCζ.

(C) BDNF stimulation promotes PKCζ interaction with Numb in GCPs. GCPs from P7 mice were treated with vehicle or BDNF for 10 minutes; lysates were precipitated and blotted with indicated antibodies.

(D) BDNF deletion impairs PKCζ interaction with Numb. Lysates from P7 wild type and bdnf^−/− cerebellum were precipitated with anti-Numb followed by blot with anti-PKCζ and anti-Numb.

(E) Numb binds active PKCζ. GST-Numb or GST immobilized on sepharose beads was incubated with purified His-tagged active PKCζ at 30 °C for 1 hour. Bound protein complex was washed and analyzed by blot with anti-His and anti-Numb, respectively. First lane, His-PKCζ alone.

(F) Time course of PKCζ activation by BDNF. GCPs from P7 were stimulated with BDNF for indicated times; lysates were blotted as indicated.

(G) Inhibition of TrkB kinase blocks BDNF-induced PKCζ activation. GCPs were treated with K252a for 30 minutes prior to BDNF stimulation; lysates blotted as indicated.

(H) Numb is required for BDNF-induced aPKC activation. GCPs from P7 Math1-Cre/Numb^flx/flx/Numbl^flx/flx or control littermates (Numb^flx/flx/Numbl^flx/flx) were treated with BDNF or vehicle. Lysates were blotted with indicated antibodies.

(I) Inhibition of PKCζ activity impairs BDNF-induced GCP chemotaxis. GCPs from P6 were treated with 10 μM chelerythrine (CHT) or vehicle and a BDNF chemotaxis assay was performed using Boyden chamber (*, P<0.05; **, P< 0.001; n=3).

(J) Inhibition of aPKC activity by CHT prevents BDNF-induced Numb polarization in GCPs. For each condition (uniform and gradient BDNF), the number of GCPs with Numb polarization toward the plug (BDNF source) was normalized to that of GCPs with Numb polarization away from the plug (**, P<0.001; n = 3).

(K) Inhibition of aPKC activity by CHT prevents BDNF-induced TrkB polarization in GCPs. For each condition (uniform and gradient BDNF), the number of GCPs with TrkB polarization toward the agarose plug (BDNF source) was normalized to that of GCPs with TrkB polarization away from the agarose plug (**, P<0.01; n = 3).