Figure 2. PKCζ is required for axon growth inhibition. A, RNAi-mediated suppression of endogenous PKCζ. Rat CGNs were transfected with the indicated amounts of either scrambled or PKCζ-targeting siRNA. Cell lysates were analyzed by SDS-PAGE and immunoblotting with anti-PKCζ antibody. The histogram shows densitometric quantitation of the PKCζ knockdown. A′, HT22 cells were transfected with HA-PKCζ and the indication siRNAs. The levels of HA-PKCζ in lysates were detected by immunoblotting with anti-HA antibody. B, PKCζ phosphorylated at T410 (p-PKCζ) was detected by immunoblotting with specific anti-phospho-PKCζ antibody. Bath application of soluble NG2 (top) or growth on NG2 coated substrates (bottom) induced the phosphorylation of PKCζ in CGNs. The histogram shows the fold increase in phospho-PKCζ ± SEM relative to control cells after normalization to total PKCζ (n = 3–6; *p < 0.05, Student's t test). C, PKCζ phosphorylated at Thr410 in HT22 cells was detected by immunoblotting. Bath application of soluble NG2 (10 μg/ml) induced the phosphorylation of PKCζ in HT22 cells. The histogram shows the mean increase in phospho-PKCζ ± SEM [n = 3; normalized to control; *not significant (NS); **p < 0.05, Students' t test]. D, Rat CGNs were transfected with FLAG-PKCζ, and soluble NG2 (10 μg/ml) was bath applied for the indicated times. PKCζ kinase activity was measured as described in Materials and Methods (n = 3; *p < 0.01; **p < 0.05, Students' t test). E, The effect of PKCζ siRNA on axon growth inhibition. Thirty picomoles of scrambled control or PKCζ-targeted siRNA along with 1.0 μg of dTomato plasmid (pCAGJC-dTomato) were transfected into CGNs. The histogram shows mean neurite lengths ± SEM (n = 1 in scrambled siRNA experiment; n = 3 in control and PKCζ siRNA experiments; *p < 0.001, Dunn's post test). F, The effect of phosphomimetic PKCζ on axon growth. One microgram of indicated PKCζ expression plasmids along with 1 μg of dTomato plasmid were transfected into CGNs. The histogram shows average neurite lengths ± SEM (n = 3; *p < 0.001, Dunn's post test). G, The effect of kinase-dead (KD) PKCζ in axon growth inhibition. Approximately 1–2 μg of indicated PKCζ expression plasmids along with 1 μg of dTomato plasmid was transfected into CGNs. For the histogram, n = 3. *p < 0.005 (Dunn's post test). Scale bars: 50 μm.