Journal: The FASEB Journal

Article Title: Increased levels of Aβ42 decrease the lifespan of ob/ob mice with dysregulation of microglia and astrocytes

doi: 10.1096/fj.201901028rr

Figure Lengend Snippet: FIGURE 3 Levels of neuronal markers were not altered in 18-month-old AppNL-F/wt knock-in; ob/ob mice. Levels of NF-H (A), SYT1 (B), PSD95 (C), and phospho-tau (D) were assessed using ELISAs and compared among each genotype after adjusting for sex (n = 10-44 mice/group). Standard curve of ELISAs of NF-H (E), SYT1 (F), PSD95 (G), and phospho-tau (H) with sample range as shown in red line in a representative assay. A-D, Data are presented as adjusted means ± standard errors of the means and were compared among each genotype using Tukey's HSD test. APP KI, AppNL-F/wt knock-in; APP KI ob/ob, AppNL-F/wt knock-in; ob/ob mice; NS, not significant; WT, wild type

Article Snippet: Recombinant human SYT1 proteins (Origene) were used as standards.

Techniques: Knock-In

Journal: The FASEB Journal

Article Title: Increased levels of Aβ42 decrease the lifespan of ob/ob mice with dysregulation of microglia and astrocytes

doi: 10.1096/fj.201901028rr

Figure Lengend Snippet: FIGURE 5 Levels of neuronal and glial markers in young (6-month-old) AppNL-F/wt knock-in; ob/ob mice. A, Body weight of young mice were compared among each genotype after adjusting for sex (n = 8-17 mice/group). B-F, Levels of NF-H (B), SYT1 (C), PSD95 (D), CD11b (E), and GFAP (F) in the brains of young mice were compared among each genotype after adjusting for sex (n = 9-17 mice/group). Data are presented as adjusted means ± standard errors of the means. *P < .05, **P < .01, and ***P < .001 for the comparisons among each genotype using Tukey's HSD test. APP KI, AppNL-F/wt knock-in; APP KI ob/ob, AppNL-F/wt knock-in; ob/ob mice; NS, not significant; WT, wild type

Article Snippet: Recombinant human SYT1 proteins (Origene) were used as standards.

Techniques: Knock-In

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: 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

Journal: Theranostics

Article Title: ANKRD22, a novel tumor microenvironment-induced mitochondrial protein promotes metabolic reprogramming of colorectal cancer cells

doi: 10.7150/thno.37472

Figure Lengend Snippet: Synergistic effect of ANKRD22 and E-Syt1 results in the abnormal accumulation of lipids in mitochondria of CCICs. (A) Lipid metabolism-related proteins of the pull-down products of ANKRD22-overexpressing cells cultured in conventional 2D condition or enriched by organoid culture. (B) Expression of E-Syt1 in the normal colorectal epithelium and CRC epithelium by IHC. (C) Co-IP verification of the interaction between ANKRD22 and E-Syt1 in conventional 2D-cultured or organoid-cultured RKO cells with Halo-ANKRD22 overexpression. (D-E) Co-IP verification of the interaction between ANKRD22 and truncated E-Syt1s. Vectors encoding different regions of E-Syt1 were transfected into SGC-7901 cells that stably expressed Halo-ANKRD22 and performed Co-IP according to the Halo-tag pull-down protocol; the pull-down eluent of SGC-7901 cells was used as a negative control. (F) Co-IP verification of the interaction between ANT2 and E-Syt1. Flag-SLC25A5 /pcDNA3.1(-) or pcDNA3.1(-) plasmids were transfected into E-Syt1-overexpressing 293T cells. Co-IP assay was conducted after 48 hours. (G) Influence of SLC25A5 knockdown on the interaction between ANKRD22 and E-Syt1. SLC25A5 -knockdown RKO cells and scrambled shRNA-infected RKO cells were infected with Halo-ANKRD22 lentivirus, and the pull-down assay was performed; the pull-down eluent of scrambled shRNA-infected RKO cells was used as a negative control. (H) Influence of E-Syt1 knockdown on the interaction between ANKRD22 and ANT2. ESYT1 -knockdown RKO cells and scrambled shRNA-infected RKO cells were infected with Halo-ANKRD22 lentivirus and the pull-down assay was performed; the pull-down eluent of scrambled shRNA-infected RKO cells was used as a negative control. (I-K) Effect of mitochondria-localized E-Syt1 on mitochondrial morphology. An E-Syt1 sequence fused with a mitochondrial localization sequence at the C-terminus was transfected into ANKRD22-overexpressing 293T cells and observed by electron microscopy. The percentage of vacuole-like and crista-reduced mitochondria in each cell was calculated (n=6). (L) Detection of E-Syt1 in the mitochondria of organoid-cultured RKO cells (Halo-ANKRD22 overexpression vs control) by WB. β-Tubulin and VDAC1 were internal references for the cytoplasmic (C) and mitochondrial (M) fractions, respectively. (M) Effect of ANKRD22 and E-Syt1 on cytoplasmic Ca 2+ level. Fluo-4 staining was used to detect the effect of ANKRD22 or synergistic effect of ANKRD22 and E-Syt1 on the cytoplasmic Ca 2+ level of SGC-7901 cell in vitro . Three replicates were performed for each group. The fluorescence intensity of FITC was detected by FCM. The data were analyzed by Student's t -test and are presented as mean±SD, *** p <0.001. (N) Effect of Ankrd22 knockout on cytoplasmic Ca 2+ level. Primary colorectal cells of Ankrd22 -/- mice (n=3) and wild-type C57BL/6 mice (n=3) were stained with fluo-4. The average fluorescence intensity was analyzed by Student's t -test, and data are presented as mean±SD, * p < 0.05.

Article Snippet: The following primary antibodies were used in this study: β-Tubulin (HuaBio, China), p38 MAPK (Cell Signaling Technology, USA), phos-p38 MAPK (Thr180/Tyr182) (Cell Signaling Technology), phos-SAPK/JNK (Thr183/Tyr185) (Cell Signaling Technology), MAX (Abcam, UK), VDAC1 (Abcam), MDH2 (Abcam), Histone H3 (Cell Signaling Technology), AMPK (Cell Signaling Technology), phos-AMPK(Thr172) (Cell Signaling Technology), phos-ACC (Ser79) (Cell Signaling Technology), PDK1 (HuaBio), ANT2 (Celling Signaling Technology), Flag Tag (HuaBio), LC3B (Cell Signaling Technology), SQSTM1/P62 (Cell Signaling Technology), E-Syt1 (Proteintech, China), DAG (LifeSpan BioSciences, USA), PIP2 (Invitrogen, USA), p53 (Cell Signaling Technology).

Techniques: Cell Culture, Expressing, Co-Immunoprecipitation Assay, Over Expression, Transfection, Stable Transfection, Negative Control, Knockdown, shRNA, Infection, Pull Down Assay, Sequencing, Electron Microscopy, Control, Staining, In Vitro, Fluorescence, Knock-Out