bic  (Alomone Labs)

Journal: Neuropsychopharmacology

Article Title: Synaptic control of DNA methylation involves activity-dependent degradation of DNMT3A1 in the nucleus

doi: 10.1038/s41386-020-0780-2

Figure Lengend Snippet: GluN2A-dependent NMDAR signaling regulates nuclear DNMT3A1 protein levels in 14–15 DIV hippocampal primary neurons. a , b Treatment with the NMDAR antagonist APV (20 μM) prevented the reduction in the nuclear levels of DNMT3A1 following synaptic stimulation by Bic/4AP for 6 h. c , d Treatment with the GluN2A inhibitor NVP-AAM077 (50 nM) prevented the reduction in the nuclear levels of DNMT3A1 following synaptic stimulation by Bic/4AP for 6 h. Two-way ANOVA followed by Bonferroni’s post hoc test. e Timeline view of hippocampal neuron treatment by Bic/4AP for 6 h with or without NVP-AAM077. f , g Quantitative immunoblotting revealed the inhibition of DNMT3A1 degradation by the use of NVP-AAM077. One-sample t-test is performed while the hypothetical value is set to 100. h , i shRNA-based knockdown of GluN2A in the presence of synaptic stimulation by Bic/4AP for 6 h prevented the DNMT3A1 degradation. j , k Treatment with the GluN2B subunit inhibitor ifenprodil (10 μM) in the presence of synaptic stimulation with Bic/4AP for 6 h did not prevent the reduction in the nuclear levels of Dnmt3A1. Scale bars, 20 μm. Two-way ANOVA followed by Bonferroni’s post hoc test. *** p

Article Snippet: Cortical neurons were treated with 1 μM tetrodotoxin (TTX, Alomone Labs, Jerusalem, Israel) for 12 h, media was washed-out and Bic/4AP were applied for 6 h at 21 DIV.

Techniques: Inhibition, shRNA

Journal: Neuropsychopharmacology

Article Title: Synaptic control of DNA methylation involves activity-dependent degradation of DNMT3A1 in the nucleus

doi: 10.1038/s41386-020-0780-2

Figure Lengend Snippet: DNMT3A1 degradation is a neddylation-dependent process. 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

Article Snippet: Cortical neurons were treated with 1 μM tetrodotoxin (TTX, Alomone Labs, Jerusalem, Israel) for 12 h, media was washed-out and Bic/4AP were applied for 6 h at 21 DIV.

Techniques: Staining, Immunoprecipitation, Immunocytochemistry, Blocking Assay, Immunofluorescence, Methylation, Activation Assay

Journal: Neuropsychopharmacology

Article Title: Synaptic control of DNA methylation involves activity-dependent degradation of DNMT3A1 in the nucleus

doi: 10.1038/s41386-020-0780-2

Figure Lengend Snippet: Synaptic activity regulates DNMT3A1 protein levels in neurons. a Nuclear DNMT3A1 immunofluorescence is prominent in MAP2-positive neurons but much less in GFAP-positive astrocytes at 15 DIV hippocampal cultures. Scale bars are 20 μm. b , c Downregulation of DNMT3A1 protein levels in 14–15 DIV hippocampal primary neurons following treatment with Bic/4AP for 1 h, 3 h or 6 h as evidenced by quantitative immunocytochemistry. Scale bar is 20 μm. Unpaired Student’s t -test ** p

Article Snippet: Cortical neurons were treated with 1 μM tetrodotoxin (TTX, Alomone Labs, Jerusalem, Israel) for 12 h, media was washed-out and Bic/4AP were applied for 6 h at 21 DIV.

Techniques: Activity Assay, Immunofluorescence, Immunocytochemistry

Journal: The EMBO Journal

Article Title: Fxr1 regulates sleep and synaptic homeostasis

doi: 10.15252/embj.2019103864

Figure Lengend Snippet: Fxr1 protein expression is decreased during homeostatic synaptic upscaling Western blot analysis of Fxr1 during (A), TTX (48 h treatment) induced upscaling ( n = 6 in each condition) and (B), BIC (48 h treatment) induced downscaling ( n = 8 in each condition) of primary postnatal cortical cultures. Student's t ‐test * P

Article Snippet: To induce upscaling or downscaling, cells were incubated in the presence of 1 μM TTX or 50 μM BIC (Alomone Labs, Jerusalem, Israel) or vehicle, respectively, from DIV12 to DIV14.

Techniques: Expressing, Western Blot

Journal: The EMBO Journal

Article Title: Fxr1 regulates sleep and synaptic homeostasis

doi: 10.15252/embj.2019103864

Figure Lengend Snippet: Expression of GluA2 during synaptic scaling Quantification of the % of infection by (A) AAV1 Syn GFP ( n = 3) or (B) AAV1 Syn GFP‐Fxr1 ( n = 3). Surface expression of GluA2 during upscaling (Veh n = 28, TTX n = 29). Expression of total GluA2 during upscaling (Veh n = 7, TTX n = 6). Surface expression of GluA2 during downscaling (Veh n = 18, BIC n = 18). Expression of total GluA2 during downscaling (Veh n = 4, BIC n = 4). Data information: Error bars are SEM.

Article Snippet: To induce upscaling or downscaling, cells were incubated in the presence of 1 μM TTX or 50 μM BIC (Alomone Labs, Jerusalem, Israel) or vehicle, respectively, from DIV12 to DIV14.

Techniques: Expressing, Infection

Journal: bioRxiv

Article Title: Fxr1 regulates sleep and synaptic homeostasis

doi: 10.1101/709345

Figure Lengend Snippet: Fxr1 protein expression is decreased during homeostatic synaptic upscaling. Western blot analysis of Fxr1 during a , TTX induced upscaling (n=6 in each condition) and b , BIC induced downscaling (n=4 in each condition) of primary postnatal cortical cultures. Student’s T-test *p

Article Snippet: To induce upscaling or downscaling cell were incubated in the presence of 1µM TTX or 50µM BIC (Alomone Labs, Jerusalem, Israel) or vehicle respectively from DIV12 to DIV14.

Techniques: Expressing, Western Blot