Journal: Journal of Biological Chemistry

Article Title: Wnt-5a/JNK Signaling Promotes the Clustering of PSD-95 in Hippocampal Neurons

doi: 10.1074/jbc.m808986200

Figure Lengend Snippet: FIGURE 7. Wnt-5a induces the clustering of PSD-95 through activation of the Wnt/JNK non-canonical pathway. A, representative neurite images of hippocampal neurons treated with or without Wnt-5a for 1 h co-incubated or not with SP600125 (SP). Neurons were treated with anisomycin for 1 h as a control for JNK activation. B, quantitative immunofluorescence of PSD-95 clusters/100 m of neurite exposed to the different treatments (n 3). C, representative neurite images of hippocampal neurons treated with different concentrations of the TAT-TI-JIP peptide in the presence of Wnt-5a or pcDNA. D, quantitative immunofluorescence of PSD-95 clusters number/100 m of neurite exposed to treatments indicated in C (n 3). *, p 0.05.

Article Snippet: Reagents—Formylated hexapeptide was obtained from GenemedSynthesis, Inc. (South SanFrancisco,CA); fumagillin, anisomycin, palmitate, and 2-bromopalmitate were from Sigma; bisindolylmaleimide-X (BSD-X), SP600125, KN-93, and TATTI-JIP-(153–163) were from Calbiochem; rabbit anti-synapsin-1, mouse anti- -catenin, mouse anti-transferrin, rabbit anti- -tubulin, rabbit anti-GAPDH, mouse anti-N-cadherin, mouse anti-CAMKII, goat anti-PSD-95, and goat anti-Wnt-5a were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); mouse anti-PSD-95 was from the University of California/ NINDS/NIMH, National Institutes of Health (Davis, CA); rabbit anti-JNK and rabbit anti-phospho-JNK were from Cell Signaling Technology (Beverly, MA); antibodies and secondary antibodies labeled with 488Alexa, 543Alexa, or 633Alexa were from Affinity Bio Reagents Inc. (Golden, CO); and phalloidin labeledwith 633Alexawas fromMolecular Probes, Inc. (Eugene, OR).

Techniques: Activation Assay, Incubation, Control, Immunofluorescence

Journal: The Journal of pharmacology and experimental therapeutics

Article Title: Downregulation of TrkB Expression and Signaling by Valproic Acid and Other Histone Deacetylase Inhibitors.

doi: 10.1124/jpet.119.258129

Figure Lengend Snippet: Fig. 1. VPA downregulates TrkB expression in RA- differentiated neuroblastoma cells. (A) SH-SY5Y cells were incubated for 24 hours with either vehicle or 1 mM VPA, and the expression of full length (TrkB-FL) and truncated (TrkB-T) isoforms of TrkB was analyzed by Western blot and normalized to actin levels. Values are the mean 6 S.E.M. of six independent experiments. (B) Quantitative real-time RT-PCR analysis of TrkB mRNA in SH-SY5Y cells treated for 24 hours with either vehicle or 1 mM VPA. Values are the mean 6 S.E.M. of three independent determinations. (C–E) SH-SY5Y cells were treated as in (A), and the expression of TrkA (C), TrkC (D), and p75NTR (E) was determined in cell lysates. (F and G) Kelly (F) and LAN-1 (G) cells were treated for 24 hours with either vehicle or VPA (1 mM). Cell lysates were then analyzed for TrkB expression. Values are the mean 6 S.E.M. of four (Kelly) and seven (LAN-1) experiments. *P , 0.05; ***P , 0.001 vs. control (vehicle) by Student’s t test.

Article Snippet: Membranes were blocked, washed, and incubated overnight at 4°C with one of the following primary antibodies: TrkB (cat. no. 4603; Cell Signaling Technology, Danvers, MA) (1:1000), TrkA (cat. no. 2508; Cell Signaling Technology) (1:1000), TrkC (cat. no. 3376; Cell Signaling Technology) (1:1000), phospho-TrkA (Tyr674/675)/TrkB (Tyr706/ 707) (cat. no. 4621; Cell Signaling Technology) (1:1000), p75NTR (cat. no. 8238; Cell Signaling Technology) (1:1000), synapsin-1 (cat. no. 5297; Cell Signaling Technology) (1:2000), pan-cadherin (cat. no. 4073; Cell Signaling Technology) (1:1000), phospho-S6 ribosomal protein (Ser235/ 236) (cat. no. 4856; Cell Signaling Technology), S6 ribosomal protein (cat. no. sc-74459; Santa Cruz Biotechnology) (1:1000), phospho-Akt (Thr308) (cat. no. 2965; Cell Signaling Technology) (1:1000), Akt1/2/3 (cat. no. sc-8312; Santa Cruz Biotechnology) (1:1000), phospho-glycogen synthase kinase (GSK)-3b (Ser9) (cat, no. 9356; Cell Signaling Technology) (1:1000), GSK-3b (cat no. sc-9166; Santa Cruz Biotechnology) (1:2000), extracellular signal-regulated kinases 1 and 2 (ERK1/2) (cat no. 9102; Cell Signaling Technology) (1:1000), phospho-ERK1 (Thr202/Tyr204)/ERK2 (Thr185/Tyr187) (cat. no. RA15002; Neuromics, Northfield, MN) (1:15,000), phospholipase Cg1 (PLCg1) (cat no. 2822; Cell Signaling Technology) (1:1000), phospho-PLCg1 (Tyr783) (cat. no. 2821; Cell Signaling Technology) (1:1000), cleaved-poly(ADPribose) polymerase (PARP) (Asp214) (cat. no. 5625; Cell Signaling Technology) (1:1000), PARP (cat. no. 9542; Cell Signaling Technology) (1:1000), enhancer of zeste homolog 2 (EZH2) (cat. no. 5246; Cell Signaling Technology) (1:2000), RUNX3/AML2 (cat. no. 9647; Cell Signaling Technology) (1:1000), postsynaptic density 95 (PSD95) (cat. no. 3450; Cell Signaling Technology) (1:2000), GluR1 (cat no.2040; Tocris Cookson Ltd., Avonmouth, UK) (1:1000), HDAC1 (cat. no. sc-81598; Santa Cruz Biotechnology) (1:5000), histone H3 (cat. no. GTX122148; GeneTex Inc., Irvine, CA) (1:2000), histone H3 (acetyl-Lys9/Lys14) (cat no. GTX122648; GeneTex Inc.), (1:1000), MAP2 (cat. no. 188011; Synaptic Systems GmbH, Gottingen, Germany) (1:1000), actin (cat no. A2066; Sigma-Aldrich) (1:3000), actin (cat. no. A5441; Sigma-Aldrich) (1:20,000), andglyceraldehyde 3-phosphate dehydrogenase (GAPDH) (cat. no. 247002; Synaptic Systems GmbH) (1:1000).

Techniques: Expressing, Incubation, Western Blot, Quantitative RT-PCR, Control

Journal: The Journal of pharmacology and experimental therapeutics

Article Title: Downregulation of TrkB Expression and Signaling by Valproic Acid and Other Histone Deacetylase Inhibitors.

doi: 10.1124/jpet.119.258129

Figure Lengend Snippet: Fig. 1. VPA downregulates TrkB expression in RA- differentiated neuroblastoma cells. (A) SH-SY5Y cells were incubated for 24 hours with either vehicle or 1 mM VPA, and the expression of full length (TrkB-FL) and truncated (TrkB-T) isoforms of TrkB was analyzed by Western blot and normalized to actin levels. Values are the mean 6 S.E.M. of six independent experiments. (B) Quantitative real-time RT-PCR analysis of TrkB mRNA in SH-SY5Y cells treated for 24 hours with either vehicle or 1 mM VPA. Values are the mean 6 S.E.M. of three independent determinations. (C–E) SH-SY5Y cells were treated as in (A), and the expression of TrkA (C), TrkC (D), and p75NTR (E) was determined in cell lysates. (F and G) Kelly (F) and LAN-1 (G) cells were treated for 24 hours with either vehicle or VPA (1 mM). Cell lysates were then analyzed for TrkB expression. Values are the mean 6 S.E.M. of four (Kelly) and seven (LAN-1) experiments. *P , 0.05; ***P , 0.001 vs. control (vehicle) by Student’s t test.

Article Snippet: Membranes were blocked, washed, and incubated overnight at 4°C with one of the following primary antibodies: TrkB (cat. no. 4603; Cell Signaling Technology, Danvers, MA) (1:1000), TrkA (cat. no. 2508; Cell Signaling Technology) (1:1000), TrkC (cat. no. 3376; Cell Signaling Technology) (1:1000), phospho-TrkA (Tyr674/675)/TrkB (Tyr706/ 707) (cat. no. 4621; Cell Signaling Technology) (1:1000), p75NTR (cat. no. 8238; Cell Signaling Technology) (1:1000), synapsin-1 (cat. no. 5297; Cell Signaling Technology) (1:2000), pan-cadherin (cat. no. 4073; Cell Signaling Technology) (1:1000), phospho-S6 ribosomal protein (Ser235/ 236) (cat. no. 4856; Cell Signaling Technology), S6 ribosomal protein (cat. no. sc-74459; Santa Cruz Biotechnology) (1:1000), phospho-Akt (Thr308) (cat. no. 2965; Cell Signaling Technology) (1:1000), Akt1/2/3 (cat. no. sc-8312; Santa Cruz Biotechnology) (1:1000), phospho-glycogen synthase kinase (GSK)-3b (Ser9) (cat, no. 9356; Cell Signaling Technology) (1:1000), GSK-3b (cat no. sc-9166; Santa Cruz Biotechnology) (1:2000), extracellular signal-regulated kinases 1 and 2 (ERK1/2) (cat no. 9102; Cell Signaling Technology) (1:1000), phospho-ERK1 (Thr202/Tyr204)/ERK2 (Thr185/Tyr187) (cat. no. RA15002; Neuromics, Northfield, MN) (1:15,000), phospholipase Cg1 (PLCg1) (cat no. 2822; Cell Signaling Technology) (1:1000), phospho-PLCg1 (Tyr783) (cat. no. 2821; Cell Signaling Technology) (1:1000), cleaved-poly(ADPribose) polymerase (PARP) (Asp214) (cat. no. 5625; Cell Signaling Technology) (1:1000), PARP (cat. no. 9542; Cell Signaling Technology) (1:1000), enhancer of zeste homolog 2 (EZH2) (cat. no. 5246; Cell Signaling Technology) (1:2000), RUNX3/AML2 (cat. no. 9647; Cell Signaling Technology) (1:1000), postsynaptic density 95 (PSD95) (cat. no. 3450; Cell Signaling Technology) (1:2000), GluR1 (cat no.2040; Tocris Cookson Ltd., Avonmouth, UK) (1:1000), HDAC1 (cat. no. sc-81598; Santa Cruz Biotechnology) (1:5000), histone H3 (cat. no. GTX122148; GeneTex Inc., Irvine, CA) (1:2000), histone H3 (acetyl-Lys9/Lys14) (cat no. GTX122648; GeneTex Inc.), (1:1000), MAP2 (cat. no. 188011; Synaptic Systems GmbH, Gottingen, Germany) (1:1000), actin (cat no. A2066; Sigma-Aldrich) (1:3000), actin (cat. no. A5441; Sigma-Aldrich) (1:20,000), andglyceraldehyde 3-phosphate dehydrogenase (GAPDH) (cat. no. 247002; Synaptic Systems GmbH) (1:1000).

Techniques: Expressing, Incubation, Western Blot, Quantitative RT-PCR, Control