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Fig. 3 | Cell Communication and Signaling

Fig. 3

From: Ethanol-activated CaMKII signaling induces neuronal apoptosis through Drp1-mediated excessive mitochondrial fission and JNK1-dependent NLRP3 inflammasome activation

Fig. 3

Ethanol-induced CaMKII activation promotes translocation of Drp1 to the mitochondria. a SK-N-MC cells were incubated EtOH (200 mM) for 12 h and then harvested. CaMKII was immunoprecipitated with an anti-CaM, anti-CaMKII antibodies (left). The expression of CaM, CaMKII, and β-Actin in total cell lysates is shown (right). n = 3. b Cells were treated with EtOH for various times (0–24 h). CaMKII and p-CaMKII (Thr 286) were analyzed by western blot. β-Actin was used as a loading control. n = 4. c Cells were pretreated with MK-801 (10 μM) for 30 min prior to EtOH treatment for 24 h. p-CaMKII (Thr 286) and CaMKII were detected by western blot. n = 4. d Cells were pretreated with KN-93 (1 μM) for 30 min prior to EtOH treatment for 24 h. Drp1 and p-Drp1 (Ser616) were detected by western blot. β-Actin was used as a loading control. n = 4. e Cells were incubated EtOH (200 mM) for 24 h and then harvested. CaMKII was immunoprecipitated with anti-CaMKII and anti-Drp1 antibodies (left). The expression of CaMKII, Drp1 and β-Actin in total cell lysates is shown (right). n = 3. f Cells were pretreated with KN-93 (1 μM) for 30 min prior to EtOH treatment for 48 h. Mitochondrial and cytosolic fractions were mentioned in methods. Drp1 was detected by western blot. Anti-TOMM20 was used as mitochondria marker. n = 3. g Cells were pre-treated with KN-93 (1 μM) for 30 min prior to EtOH treatment for 24 h and immunostained with Drp1 antibody and Mitotracker™. Co-localization of Drp1 (green) and Mitotracker™ (red) was visualized with SRRF imaging system. Scale bars are 8 μm (magnification, × 1,000). n = 4. All data are presented as a mean ± S.E.M. All blot and immunofluorescence images shown are representative. *p < 0.05 versus control, #p < 0.05 versus EtOH.

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