Fig. 3 | Nature Communications

Fig. 3

From: CaMKII-mediated Beclin 1 phosphorylation regulates autophagy that promotes degradation of Id and neuroblastoma cell differentiation

Fig. 3The alternative text for this image may have been generated using AI.

CaMKII induces autophagy through phosphorylation of Beclin 1 at Ser90 and the subsequent ubiquitination at Lys117. a Dissociation of the Bcl-2-Beclin 1 Complex. SK-N-SH cells treated with 6 μM ionomycin or 100 nM EB1089 were lysed and incubated with anti-Beclin 1 or normal mouse IgG antibodies prior to western blot analysis. The resulting immunoprecipitates were subjected to western blot analysis using the indicated antibodies. TCL, whole-cell lysate. b Autophagy induced by ionomycin in neuroblastoma cells. Both cell lines were treated with 6 μM ionomycin for the indicated periods or various concentrations of ionomycin for 24 h. The total cell lysates were analyzed for LC3 lipidation by immunoblotting. c LC3 mRNA expression was unaffected in cells treated with ionomycin or EB1089. The SK-N-SH cells were untreated or were treated with ionomycin or EB1089 of indicated concentration for 24 h. The expression level of LC3 mRNA was detected by real-time RT-PCR. The error bars represent the standard deviations (SD) calculated from three parallel experiments. d LC3 lipidation and p62 degradation in CaMKII-KD-expressing MEFs cells or control cells (Ctrl). MEFs cells transfected with a control vector or plasmids encoding His-CaMKII-KD were treated with 6 μM ionomycin for 24 h, then incubated for 2 h in the presence or absence of 100 nM Baf A1. The whole-cell extracts were subjected to western blot using the indicated antibodies. e LC3 lipidation and p62 degradation in CaMKII shRNA MEFs cells or negative control cells (NC). MEFs cells transfected with negative control or CaMKII shRNA were treated with 6 μM ionomycin for 24 h, then incubated for 2 h in the presence or absence of 100 nM Baf A1. The whole-cell extracts were subjected to western blot using the indicated antibodies. f LC3 lipidation in Beclin 1-deficient MEFs expressing WT Beclin 1 or S90A-Beclin 1. The cells were incubated in 6 μM ionomycin for 24 h, and total cell lysates were analyzed for LC3 lipidation by means of immunoblotting. g Autophagosome formation in MEFs deficient for Beclin 1. The cells were incubated in 6 μM ionomycin for 24 h and then fixed. LC3-positive puncta were shown as the means ± SEM of five random areas. **P < 0.01, Student’s t-test. Scale bar, 10 μm

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