Skip to main content
. 2008 Oct 29;28(44):11409–11420. doi: 10.1523/JNEUROSCI.2135-08.2008

Figure 4.

Figure 4.

Knockdown of PDK1 reduces ERK-dependent activation of RSK1/2. A, Freshly isolated cortical neurons were coelectroporated with two different shRNA constructs targeting PDK1 (shPDK1-1 or -2) together with an expression plasmid for FLAG-Bcl2 that was added to reduce electroporation toxicity (4 + 0.2 μg of each plasmid DNA/5 × 106 cells, respectively). The shRNA against GFP was used as a control (shGFP). After 72 h, Western blot analysis revealed that shPDK1-1 and shPDK1-2 reduced the levels of endogenous PDK1 by 70 or 80% of control, respectively. Numbers under the blot represent the ratios of endogenous PDK1 to Flag-Bcl2 levels. B, Freshly isolated cortical neurons were coelectroporated with pcDNA3-HA-ERK1, pcDNA3-HA-ERK2, and pcDNA3-FLAG-Bcl2 together with one of the following: shGFP, shERK1-1, shERK1-2, shERK2-1, and shERK2-2, as indicated (0.8 + 0.8 + 0.2 + 2.5 μg of each plasmid DNA/5 × 106 neurons, respectively). After 72 h, the levels of HA-ERK1/2 were analyzed by Western blotting with an anti-HA antibody. Numbers under the blot represent the ratios of the indicated HA-ERKs to Flag-Bcl2 levels. Reduced levels of HA-ERK1 or HA-ERK2 compared with Flag-Bcl2 indicate efficient knockdowns with shERK1 or shERK2, respectively. C, Cortical neurons were electroporated with the indicated shRNAs (3 μg plasmid DNA/10 × 106 cells). To reduce electroporation toxicity, the dominant negative p53 expression plasmid (p53-DD) was included in all samples (1 μg plasmid DNA/10 × 106 cells). After 72 h, neurons were stimulated with BDNF for 30 min and the NTK activity of the endogenous RSK1/2 was analyzed by immunocomplex kinase assay. Immunoprecipitates contained similar levels of RSK1/2 as determined by Western blotting (shown at the bottom of the graph). Depletion of PDK1 or ERK1 reduced the BDNF activation of RSK NTK. Depletion of ERK2 abolished that activation. Therefore, ERK1, ERK2, and PDK1 are required for full activation of neuronal RSK1/2 by BDNF. In A, B, similar results were obtained in two independent experiments. In C, averages of three independent experiments ±SEM are presented. *p < 0.05; **p < 0.01; ***p < 0.001; nonsignificant (NS), p > 0.05.