Journal: Journal of Biochemical and Molecular Toxicology

Article Title: Bisphenol A Treatment Impairs Synaptic Function in Human Cholinergic Neurons

doi: 10.1002/jbt.70558

Figure Lengend Snippet: Densitometric analysis of PSD95, SYT, and SYP protein levels of CTRL and BPA‐treated cells. The level of ACTIN protein was used as a loading control for protein normalization in western blot analysis. For both CTRL and BPA‐treated samples, each lane represents an independent biological replicate of the experiment. Values are reported as mean ± SEM. * p ≤ 0.05, ** p ≤ 0.01. Uncropped gels are provided in the Figure .

Article Snippet: Following blocking, the membranes were incubated overnight, at 4°C with primary antibodies against SYP (AB9272; Merk‐Millipore; dilution 1:80,000), SYT (#14558; Cell Signaling Technology; dilution 1:1000), PSD‐95 (#2507; Cell Signaling Technology; dilution 1:1000), PARP‐1 (AB‐83632; Immunological Science; dilution 1:1000), and CASP‐3 (sc‐271028; Santa Cruz; dilution 1:1000).

Techniques: Control, Western Blot

Journal: Neural Plasticity

Article Title: Brain-Specific SNAP-25 Deletion Leads to Elevated Extracellular Glutamate Level and Schizophrenia-Like Behavior in Mice

doi: 10.1155/2017/4526417

Figure Lengend Snippet: Alteration of expression pattern of SNARE-related proteins. Representative western blot (left) and densitometric analysis (right) of proteins in the cytosolic (a) and membrane (b and c) fractions prepared from mouse cerebral cortex ( n = 3 per group). ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 compared with control littermates. P-SYT: phosphorylated synaptotagmin-1.

Article Snippet: Then, the lysates were separated by SDS–PAGE and probed with specific antibodies: SNAP-25 (Abcam, ab66066), SNAP-23 (Abcam, ab3340), syntaxin (Santa Cruz, sc-12736), Vamp2 (Abcam, ab6276), Munc-18 (SYSY, 116002), Phospho-Synaptotagmin (R&D Systems, PPS085), β -ACTIN (Abcam, ab6276), TUBULIN (Abcam, ab15246), and Na/K ATPase (Millipore, 05-369).

Techniques: Expressing, Western Blot, Membrane, Control

Journal: Journal of Neuroscience

Article Title: Convergence of Presenilin- and Tau-Mediated Pathways on Axonal Trafficking and Neuronal Function

doi: 10.1523/jneurosci.1964-10.2010

Figure Lengend Snippet: Figure 3. Decreased synaptosomal BDNF levels and Erk1/2 activation in the hippocampus of 6-month-old PS cDKO;WtTau animals. A, Representative Western blots of total BDNF and BDNF from synaptosome fractions (BDNF). B, Quantification of nor- malized BDNF band densities relative to control (PS2 KO). C, Representative Western blots of total and phospho-Erk1/2. D, Quan- tification of normalized Erk1/2 band densities relative to control. Loading controls were as follows: synaptotagmin (Syt) for synaptosomal BDNF and -tubulin for all others. *p 0.05, **p 0.01.

Article Snippet: The following antibodies were used: PHF-1, 1:500 (a generous gift from Dr. Peter Davies, Albert Einstein College of Medicine, New York), AT8, 1:500 (Pierce Endogen, Thermo Fisher Scientific), Tau 5, 1:1000 (Millipore Bioscience Research Reagents), -tubulin 1:10,000 (Sigma), APPc 1:2000 (Sigma), c-Myc (9E10) 1:1000 (Santa Cruz Biotechnology), BDNF (N-20) 1:500 (Santa Cruz Biotechnology), synaptotagmin 1:500 (a generous gift from Dr. Louis Reichardt, University of California, San Francisco, School of Medicine, San Francisco, CA), NMDAR1, 1:5000 (Millipore Bioscience Research Reagents), Erk1/2, 1:1000 (Cell Signaling Technology), phospho-Erk1/2 (Thr202/Tyr204) 1:1000 (Cell Signaling Technology), Akt 1:1000 (Cell Signaling Technology), phospho-Akt 1:1000 (Cell Signaling Technology), CaMKII 1:500 (Santa Cruz Biotechnology).

Techniques: Activation Assay, Western Blot, Control

Journal: The Journal of comparative neurology

Article Title: Three-Dimensional Ultrastructure of Flower-Spray Nerve Endings in the Rat Carotid Sinus.

doi: 10.1002/cne.25654

Figure Lengend Snippet: FIGURE 1 Whole mount preparation of the carotid sinus with double immunofluorescence for P2 × 3 (green) and Syt1 (red). (a) Low magnification view of the carotid sinus showing P2 × 3-immunoreactive flower-spray nerve endings. Syt1 immunoreactivity is shown in flower-spray endings and in the network of varicose nerve fibers. (b and c) Three-dimensional view of the basal surface of the terminal part of the flower-spray ending indicated in rectangle in Panel a. Arrows indicate thick parent axon for the ending. Panel b shows flower-spray endings could be distinguished from network of thin varicose nerve fibers with Syn1 immunoreactivity. (d–f) Higher magnification view of the rectangle in Panel a. (d) Punctate P2 × 3 immunoreactivity is shown in the terminal part of the endings. (e) Syt1 immunoreactivity is shown as smaller dots in the terminal parts. (f) The merged figure shows that P2 × 3 and Syt1 immunoreactivities are distinct from each other.

Article Snippet: A mouse monoclonal anti-Syt1 antibody (clone ASV48, MAB4364, R&D Systems, Minneapolis, MN, USA; RRID AB_2199304) was raised against the rat brain synaptic plasmamembrane (Matthew, Tsavaler, and Reichardt 1981).

Techniques: Immunofluorescence

Journal: Molecular Brain

Article Title: Prenatal activation of Toll-like receptors-3 by administration of the viral mimetic poly(I:C) changes synaptic proteins, N-methyl-D-aspartate receptors and neurogenesis markers in offspring

doi: 10.1186/1756-6606-5-22

Figure Lengend Snippet: Expression of synaptic proteins. Bar charts showing the quantified expression of (A) synaptophysin (M.W.40 kDa) (B) synaptotagmin (M.W. 57 kDa) and (C) Vesicle Associated Membrane Protein-1 (VAMP-1; synaptobrevin; M.W. 14 kDa) in the brains of P21 rat offspring after treating the mothers with poly(I:C) 10 mg/kg on days E14, E16 and E18 of gestation. The bars indicate the mean ± s.e.mean (n = 5–6) in arbitrary units of optical density (OD) expressed as the ratio of test protein to actin. Sample western blots above each chart illustrate the data obtained from animals exposed to the saline vehicle (S) or poly(I:C) (P) and show the relevant protein and the corresponding actin blot used as a housekeeping marker * P < 0.05

Article Snippet: Western blot analysis was carried out using the following primary antibodies raised against target proteins: GluN1 (mouse monoclonal, 05–432, 1 : 1000 dilution) and synaptophysin (mouse monoclonal, MAB368, 1 : 40000 dilution) (Millipore, Watford, UK); GluN2A (rabbit polyclonal, PPS012, 1 : 5000 dilution), GluN2B (rabbit polyclonal, PPS013, 1 : 5000 dilution), VAMP-1/synaptobrevin (goat polyclonal, AF4828, 1 : 10000 dilution), and synaptotagmin (mouse monoclonal, MAB43641, 1 : 5000 dilution) (R&D Systems, Abingdon, UK); PSD-95 (rabbit monoclonal, 3450, 1 : 10000 dilution) (Cell Signalling, New England Biolabs, Hitchin, UK); RhoA (mouse monoclonal, sc-418, 1 : 1000 dilution), RhoB (mouse monoclonal, sc-8048, 1 : 1000 dilution), EphA4 (rabbit polyclonal, sc-921, 1 : 5000 dilution), PCNA (mouse monoclonal, sc-56, 1 : 1000 dilution), doublecortin (goat polyclonal, sc-8066, 1 : 1000 dilution), Sox-2 (goat polyclonal, sc-17320, 1 : 500 dilution) and actin (goat polyclonal, sc-1615, 1 : 10000 dilution) (Santa Cruz, Insight Biotechnology, Wembley, UK).

Techniques: Expressing, Membrane, Western Blot, Saline, Marker

Journal: Scientific Reports

Article Title: Down-regulation of HMGB1 expression by shRNA constructs inhibits the bioactivity of urothelial carcinoma cell lines via the NF-κB pathway

doi: 10.1038/srep12807

Figure Lengend Snippet: A1, A2 and A3: The expression of NF-κB/p65 and VEGF-C in the shRNA group was lower than in the other two groups transfected with shNC plasmids or the untransfected controls (all P < 0.05). On the contrary, the expression of IκBα showed an opposite tendency, while no significant differences in the expression of NF-κB/p65, IκBα and VEGF-C in T24 cells were found between the CON and NC groups (all P > 0.05). The display of cropped gels is used to improve the clarity and conciseness of the presentation, and all the cropped gels have been run under the same experimental conditions. : The blue areas indicate nuclei stained using 4, 6-diamidino-2-phenylindole (DAPI), and the green areas indicate the nuclear translocation of NF-κB/p65 in T24 cells transfected with shNC plasmids or untransfected and cytoplasmic localization of NF-κB/p65 in cells transfected with shRNA plasmids. The results showed that knockdown of HMGB1 expression inhibited the translocation of NF-κB/p65 from the cytoplasm to the nucleus. : EMSA revealed that the DNA-binding activity of NF-κB/p65 in T24 cells was decreased by HMGB1 knockdown.

Article Snippet: The specific antibodies were Mouse HMGB1 antibody (Santa Cruz Biotechnology, Santa Cruz, USA; 1:500 dilution), Rabbit NF-κB/p65 antibody (Boster Biological Technology, Wuhan, China; 1:100 dilution), Rabbit IκBα antibody (Abcam, Cambridge, USA; 1:500 dilution), Rabbit VEGF-C antibody (Boster Biological Technology, Wuhan, China; 1:100 dilution), and Mouse GAPDH antibody (Santa Cruz Biotechnology, Santa Cruz, USA; 1:800 dilution).

Techniques: Expressing, shRNA, Transfection, Staining, Translocation Assay, Knockdown, Binding Assay, Activity Assay