Fig. 6 | Nature Communications

Fig. 6

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

Fig. 6The alternative text for this image may have been generated using AI.

TRAF-6 induced the K63-linked ubiquitination of Id-1 at the K88 residue. a CaMKII and phosphorylation of Beclin 1 Ser90 are required for K63 ubiquitination of Ids. HEK293T cells were transiently co-transfected with the indicated plasmids, treated with 6 μM ionomycin for 24 h, and then incubated with 100 nM Baf A1 for 4 h. The immunoprecipitated Myc-Ids was subjected to anti-K63-ubiquitin western blot. b Id-1 and Id-2 undergoes K63 ubiquitination. HEK293T cells were transiently transfected with HA-Ubi or HA-Ubi-K63R plasmids, treated with 6 μM ionomycin or 100 nM EB1089 for 24 h, and then incubated with 100 nM Baf A1 for 4 h. The immunoprecipitated Myc-Id-1/2 was subjected to anti-HA western blot. c TRAF-6 is required for the K63-ubiquitination of Id-1/2. HEK293T cells were transfected with the indicated plasmids for 24 h, incubated with 6 μM ionomycin or 100 nM EB1089 for 24 h, and then treated with Baf A1 for 4 h. The immunoprecipitated Myc-Id-1/2 was subjected to anti-HA western blot. d TRAF-6 is required for the degradation of Id-1/2. HEK293T cells were transfected for 24 h with the negative control (NC) or TRAF-6 shRNA and then incubated for 24 h with 6 μM ionomycin or 100 nM EB1089. The cell lysates were then analyzed by western blot. e The bHLH domain of Id-1 was required for its interaction with TRAF-6. HEK293T cells were transiently transfected with the indicated plasmids for 24 h and then treated with 6 μM ionomycin for 24 h and 100 nM Baf A1 for 2 h before lysis. The whole-cell extracts were subjected to western blotting using the indicated antibodies after immunoprecipitation. f The lysine88 residues in the bHLH domain of Id-1 is more conserved during evolution (indicated with a red background and the other two with a blue background). g The K88 residue of Id-1 was necessary to the K63-linked ubiquitination of Id-1 induced by ionomycin. SK-N-SH cells were transiently co-transfected with plasmids encoding Myc-Id-1-WT or K88R and plasmid with HA-Ubiquitin, then treated with 6 μM ionomycin for 24 h, and with Baf A1 for 4 h. The cell lysates were then analyzed by western blot after the immunoprecipitation

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