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. 2016 Dec 22;8(1):95–111. doi: 10.1016/j.stemcr.2016.11.009

Figure 3.

Figure 3

PDK1 Is Involved in HPCA-Mediated PKCα Activation and Neuronal Differentiation of NSCs

(A) NSCs were pretreated with 5 μM GSK2334470 or 10 μM BAG956 for 30 min before differentiation for 1 day. Cells were lysed and analyzed by western blotting with anti-p-PKCα, anti-PKCα, anti-p-PDK1, anti-PDK1, and anti-GAPDH.

(B and C) Cells were pretreated with 5 μM GSK 2334470 and allowed to differentiate for 1 day. mRNA levels of neuronal factors were analyzed by RT-PCR (B) and real-time RT-PCR (C). ∗p < 0.05 compared with the −bFGF control (mean ± SD; n = 3).

(D) Cells were pretreated with 5 μM GSK2334470 and induced to differentiate by withdrawal of bFGF. After differentiation for 3 days, fixed cells were immunostained with anti-TUJ1 (green) and anti-GFAP (red). Scale bar, 20 μm.

(E and F) Neurite lengths were measured and the numbers of TUJ1-positive cells and total cells were determined in randomly selected areas from at least three slides of each condition. ∗p < 0.05 compared with the −bFGF control (mean ± SD; n = 3).

(G) Cells were transfected with pMSCV-IRES-EGFP or pMSCV-IRES-EGFP-Hpca-Myc for 48 hr, and induced to differentiate for 24 hr. Immunoprecipitates from the cell lysates with anti-PDK1 were analyzed by western blotting with anti-p-PKCα, anti-PKCα, anti-PDK1, and anti-MYC.