Fig. 4.

Pyk2 regulates CREB expression and cell viability. A: time course. SH-SY5Y cells stably expressing V5-TRPM2-L were transiently transfected with shRNA targeting Pyk2, and samples for Western blotting removed from culture at 24-h intervals for 168 h. Western blotting was performed with antibodies to V5, pPyk2, Pyk2, pCREB, and CREB. Tubulin was probed to confirm equivalent loading. B: SH-SY5Y cells expressing V5-TRPM2-L were stably transfected with shRNA targeting Pyk2 or control scrambled shRNA. Western blotting was performed on lysates from untreated cells with antibodies to V5, pPyk2, Pyk2, pCREB, and total CREB and confirmed that down modulation of Pyk2 resulted in reduced pPyk2, pCREB, and CREB. A representative Western blot from one of three experiments is shown. C: expression of pPyk2, Pyk2, pCREB, CREB, and tubulin was normalized by comparison of expression in cells transfected with Pyk2 targeted shRNA to that in scrambled shRNA for each densitometry measurement in the three experiments in B. The Student’s t-test was used for analysis of differences. *P ≤ 0.05. D: down modulation of Pyk2 resulted in a significant reduction in live cell number after doxorubicin in three experiments, measured with trypan blue exclusion. Measurements were standardized to results for untreated cells for each group, and the means ± SE of six replicates from one representative experiment are shown. *P ≤ 0.05. CREB, cAMP-responsive element-binding protein; Doxo, doxorubicin; p, phosphorylated; Pyk2, proline-rich tyrosine kinase 2; TRPM2, transient receptor potential melastatin channel subfamily member 2; TRPM2-L, full-length TRPM2.