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. 2020 Jul 29;45(12):2120–2130. doi: 10.1038/s41386-020-0780-2

Fig. 3. DNMT3A1 degradation is a neddylation-dependent process.

Fig. 3

a NEDD8 is abundantly expressed in the nucleus of 16 DIV hippocampal neurons whereas weaker staining was observed at synapses. Scale bar 20 μm (top panel), 5 μm (lower panel). b Immunoprecipitation of CUL4B protein from nuclear extracts of cortical primary neurons (top panel) results in co-precipitation of DNMT3A1 (lower panel, indicated with an arrow). c, d Hippocampal primary neurons were treated with Bic/4AP for 6 h in the presence or absence of MLN4924 (5 nM). Scale bars, 20 μm. Quantitative immunocytochemistry revealed that blocking neddylation prevents DNMT3A1 degradation. e Representative immunofluorescence images of nuclear NEDD8 and total cytosine methylation (5meC) at basal conditions or after treatment with Bic/4AP for 6 h. Scale bar is 20 μm. f, g Upon synaptic activation nuclear NEDD8 and 5meC levels remained unchanged. Unpaired Student’s t-test. n.s. not significant. h, i CUL4B was immunoprecipitated from nuclear extracts of primary cortical neurons and neddylated CUL4B was quantified. Following 10 min of synaptic stimulation, the amount of neddylated CUL4B was increased. Unpaired Student’s t-test **p < 0.01. j, k shRNA knockdown of NEDD8 in hippocampal primary neurons reduced DNMT3A1 degradation following 10 min-long Bic/4AP treatment and fixation of cells 3 h after washout of the drug-containing-media. Two-way ANOVA followed by Bonferroni’s post-hoc test. ***p < 0.001, scale bar, 20 μm. Error bars present S.E.M. Sample numbers for each experimental group indicate neurons from three different culture preparations.