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. 2012 May 23;32(21):7278–7286. doi: 10.1523/JNEUROSCI.6273-11.2012

Figure 5.

Figure 5.

Fyn is activated by Lck during PC neuroprotection. A, The active form of Fyn was increased after NMDA PC. Phosphorylation of Fyn at Tyr 417 was examined by Western blot after immunoprecipitation with Fyn Ab. Total levels of Fyn in each immunoprecipitate was also determined. B, Fyn kinase activity was enhanced by NMDA PC. *p < 0.05 versus control without PC. C, NMDA-induced cytotoxicity was examined after siFyn. D, Silencing Fyn reversed PC neuroprotection. siFyn was transfected 48 h before PC, and PC was applied at 24 h before the lethal stimuli. Cytotoxicity was determined at 24 h after lethal NMDA exposure. *p < 0.05 versus NMDA lethal; #p < 0.05 versus Neg+NMDA PC+NMDA lethal. E, Fyn levels were decreased by siFyn transfection. *p < 0.05, **p < 0.01 versus Neg. F, Silencing Fyn significantly reversed OGD PC neuroprotection. siRNAs were transfected 48 h before OGD PC, and OGD PC/lethal OGD exposure was performed as described above. **p < 0.01 versus OGD lethal; #p < 0.05 versus Neg+OGD PC+OGD lethal. G, Active form of p-Fyn was enhanced by OGD PC in cortical neurons. *p < 0.05 versus CON without OGD PC. H, Fyn activation during PC was reversed by silencing Lck. siLck was transfected 48 h before PC. p-Fyn or Fyn levels were examined by Western blot after Fyn-immunoprecipitation. N, Nontargeting negative siRNA; siL, siLck. *p < 0.05 versus Neg without PC; #p < 0.05 versus Neg with PC. I, Fyn activation by PC was not observed in neurons from Lck−/− mice. J, Silencing Fyn did not influence Lck kinase activation during PC. Lck kinase activity was determined in Lck-immunoprecipitates after siFyn transfection and PC. siF, Silencing Fyn. *p < 0.05 versus Neg without PC. K, Inhibition of PKCε decreased Fyn activation during PC. εV1-2 was treated by BioPORTER before PC, and cell lysates were collected after PC for Fyn immunoprecipitation and Western blot. εV, εV1-2; Veh, vehicle control. *p < 0.05 versus vehicle control without PC; #p < 0.05 versus Vehicle+NMDA PC. A–C, E, G–K, n = 3; D, F, n = 4. All values are means ± SEM and analyzed by Student's t test.