Figure 3.

Overexpression of DLK reduces mouse ES cell numbers and the expression of Nanog. (A) The protein expression amounts in DLK overexpression mouse ES cells were similar in Day 15 and Day 20 EBs. DLK protein was detected in DLK overexpressing mouse ES cells (D3) and EBs (5th, 10th, 15th and 20th days) by Western blotting. (B) DLK kinase activity was up-regulated upon the overexpression of DLK. DLK protein precipitated with DLK antibody was aliquoted for DLK kinase activity assays and Western blotting. MBP was used as DLK substrate to evaluate DLK kinase activity. (C) The overexpression of DLK reduced relative cell numbers in ES cells. Relative cell numbers of mouse ES cells transfected with GFP or DLK plasmid were analyzed by the Alamar blue assay in 96-well plates. In the experiment, 2.0 × 10⁴ cells were transfected. (D) Overexpression of DLK slightly down regulated the expression amount of Nanog. The expression of DLK, Nanog and Oct4 proteins were analyzed by Western blotting. The error bars in the figures represent standard error of the mean (mean±SEM). P values were obtained from 2-tailed Student's t-tests (***, P < 0.0001; **, P < 0.001; *, P < 0.05).