JAK2 knockdown upregulates CDK5. (A, B) JAK2 knockdown cells were induced to differentiate into neuronal cells for three days and expression levels of CDK5 and P35 were analyzed by semi-quantitative polymerase chain reaction (qPCR) (A) and immunoblotting (B). (C) J1ES cells were induced to differentiate into neuronal cells for 2 days and then treated with SD-1029 (indicated concentration) or DMSO for 24 h. Expression levels of CDK5 and actin were analyzed by Western blot. (D) JAK2 knockdown cells were induced to differentiate into neuronal cells for 2 days and then incubated with 2.5 μM LiCl or saline for 24 h. Whole-cell lysates were prepared on day 3 for western blot analysis of CDK5, P35, and actin expression. (E) J1 ESCs were infected with lentivirus vector encoding scrambled shRNA or shCDK5 and differentiated into neurons. Knockdown efficiency was analyzed by immunoblotting after three days of differentiation culture. (F) CDK5 knockdown cells were induced to differentiate into neuronal cells for three days and then subjected to Western blot analysis of neuron specific markers. (G) JAK2 knockdown cells were induced to differentiate into neuronal cells for three days and then GST-fused Pak binding domain was used for pull-down assay to determine GTP-bound active Rac1 protein levels, while a parallel western blot analysis of total cell lysate was performed to determine total Rac1 expression level. (H) Quantitation by densitometry. Data are expressed as mean ± SD. Treatment group means were compared by Student’s t-test (*P < 0.05; **P < 0.01; ***P < 0.005).
