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: Cells

Article Title: Mitochondria-Endoplasmic Reticulum Interplay Regulates Exo-Cytosis in Human Neuroblastoma Cells

doi: 10.3390/cells11030514

Figure Lengend Snippet: Mfn2 down-regulation decreases vesicular protein levels and vesicle number, while increasing SNARE SNAP25 protein levels and SynaptopHluorin (SypHy) fluorescence. ( A ) Schematic representation of some of the proteins involved in vesicular release mechanisms ( B ) Representative immunoblots of NC siRNA or Mfn2 siRNA SH-SY5Y cell homogenates. Blots were probed with antibodies against Mfn2, Synaptophysin, Synapsin-1, Synaptotagmin-1, Neuropeptide Y, SNAP25, Syntaxin-1 and actin which was used as a loading control ( C ) Bar graph shows the amounts of protein analyzed once standardized to actin content in each sample ( n = 6–9 independent cultures) ( D ) Representative TEM pictures of NC siRNA or Mfn2 siRNA SH-SY5Y cells showing vesicles (pink) and dense core vesicles (blue). Scale bar = 500 nm. ( E ) Violin plots show quantification of number of vesicles per cell (four independent cultures, five cells analyzed per culture) ( F ) Schematic representation showing the mode of action of SypHy construct upon KCl depolarization ( G ) Representative confocal images of SH-SY5Y cells treated with NC siRNA or Mfn2 siRNA. Panels show initial fluorescence (F 0 ) and final fluorescence (F f ). Scale bar = 10 µm ( H ) Graph shows normalized SypHy fluorescence over time in NC siRNA or Mfn2 siRNA, each timepoint ( F ) was normalized to average pre-stimulation fluorescence levels (F pre ). After one minute of basal recording, cells were depolarized with 50 mM KCl ( n = 18–19 cells from 4 independent cultures). The ratiometric difference between F f /F 0 was shown to be significant p ≤ 0.0001. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: The following antibodies were used: Actin (Sigma-Aldrich, St. Louis, MO, USA, #A4700 1:2500), Actin (Sigma-Aldrich, #A5441 1:2500), LC3 (Cell Signaling Technology, Danvers, MA, USA #3868 1:1000), Mfn2 (Abcam, Cambridge, UK, #Ab56889 1:1000), Neuropeptide Y (Cell Signaling Technology, #11976T 1:500), pSer293-PDH (Merck Millipore, Burlington, MA, USA #ABS204 1:1000), PDH (Santa Cruz, Dallas, TX, USA #sc-377,092), SNAP25 (Biolegend, San Diego, CA, USA #850301 1:1000), Synapsin-1 (Cell Signaling Technology, #5297T 1:1000), Synaptophysin (Abcam,#Ab14692 1:1000), Synaptotagmin-1 (Novus Biologicals, Littleton, CO, USA #MAB4364-SP 1:500), Syntaxin-1 (Sigma-Aldrich, #S0664) 1:500), TIM23 (BD Biosciences, Franklin Lakes, NJ, USA #611223 1:1000), and VDAC1 (Abcam, #Ab14734 1:1000).

Techniques: Fluorescence, Western Blot, Control, Construct

Journal: Cells

Article Title: Mitochondria-Endoplasmic Reticulum Interplay Regulates Exo-Cytosis in Human Neuroblastoma Cells

doi: 10.3390/cells11030514

Figure Lengend Snippet: SNAP25 KD rescues Mfn2 KD-mediated vesicle marker downregulation and decreases SypHy fluorescence and exocytosis upon cellular depolarization ( A ) Representative immunoblots of NC siRNA, Mfn2 siRNA, SNAP25 siRNA and Mfn2+SNAP25 siRNA SH-SY5Y cells. Blots were probed with antibodies against Mfn2, synaptophysin and actin which was used as a loading control. ( B ) Bar graph shows the amounts of synaptophysin protein once standardized to actin content in each sample ( n = 8 independent cultures) ( C ) Representative confocal images of Mfn2 siRNA or Mfn2+SNAP25 siRNA SH-SY5Y cells. Panels show initial fluorescence (F 0 ) and final fluorescence (F f ). Scale bar = 10 µm ( D ) Graph shows normalized SypHy fluorescence over time in NC siRNA, Mfn2 siRNA, SNAP25 siRNA and Mfn2+SNAP25 siRNA, each timepoint (F) was normalized to average pre-stimulation fluorescence levels (F pre ). After one minute of basal recording, cells were depolarized with 50 mM KCl ( n = 11–22 cells from 5 independent cultures). The ratiometric difference between Mfn2 siRNA and Mfn2+SNAP25 siRNA F f /F 0 was shown to be significant p ≤ 0.05. Data shown as mean ± SEM. * p ≤ 0.05, *** p ≤ 0.001.

Article Snippet: The following antibodies were used: Actin (Sigma-Aldrich, St. Louis, MO, USA, #A4700 1:2500), Actin (Sigma-Aldrich, #A5441 1:2500), LC3 (Cell Signaling Technology, Danvers, MA, USA #3868 1:1000), Mfn2 (Abcam, Cambridge, UK, #Ab56889 1:1000), Neuropeptide Y (Cell Signaling Technology, #11976T 1:500), pSer293-PDH (Merck Millipore, Burlington, MA, USA #ABS204 1:1000), PDH (Santa Cruz, Dallas, TX, USA #sc-377,092), SNAP25 (Biolegend, San Diego, CA, USA #850301 1:1000), Synapsin-1 (Cell Signaling Technology, #5297T 1:1000), Synaptophysin (Abcam,#Ab14692 1:1000), Synaptotagmin-1 (Novus Biologicals, Littleton, CO, USA #MAB4364-SP 1:500), Syntaxin-1 (Sigma-Aldrich, #S0664) 1:500), TIM23 (BD Biosciences, Franklin Lakes, NJ, USA #611223 1:1000), and VDAC1 (Abcam, #Ab14734 1:1000).

Techniques: Marker, Fluorescence, Western Blot, Control

Journal: Cells

Article Title: Mitochondria-Endoplasmic Reticulum Interplay Regulates Exo-Cytosis in Human Neuroblastoma Cells

doi: 10.3390/cells11030514

Figure Lengend Snippet: Inhibition of ER to mitochondria Ca 2+ shuttling abrogates Mfn2-dependent down-regulation of synaptophysin levels and increased exocytosis ( A ) Schematic representation showing mode of action of Xestospongin C (XeC) and MCUi4 at MERCS ( B ) Representative immunoblots of NC siRNA or Mfn2 siRNA SH-SY5Y cells treated or 6 h with DMSO or 1 µM XeC. Blots were probed with antibodies against Mfn2, synaptophysin and actin, which was used as a loading control. ( C ) Bar graph shows the amounts of synaptophysin protein once standardized to actin content in each sample ( n = 5 independent cultures) ( D ) Representative confocal images of Mfn2 siRNA SH-SY5Y cells treated with DMSO or XeC 1 µM for 6 h. Panels show initial fluorescence (F 0 ) and final fluorescence (F f ). Scale bar = 10 µm ( E ) Graph shows normalized SypHy fluorescence over time in Mfn2 siRNA treated with DMSO or XeC, each timepoint ( F ) was normalized to average pre stimulation fluorescence levels (F pre ). After one minute of basal recording, cells were depolarized with 50 mM KCl ( n = 24–27 cells from four independent cultures). Ratiometric difference between F f /F 0 was shown to be significant p ≤ 0.01. ( F ) Representative immunoblots of NC siRNA or Mfn2 siRNA SH-SY5Y cells treated for 6h with DMSO or 1 µM MCUi4. Blots were probed with antibodies against Mfn2, synaptophysin and actin which was used as a loading control. ( G ) Bar graph shows the amounts of synaptophysin protein once standardized to actin content in each sample ( n = 5 independent cultures). ( H ) Representative confocal images of Mfn2 siRNA SH-SY5Y cells treated with DMSO or MCUi41 µM for 6 h. Panels show initial fluorescence (F 0 ) and final fluorescence (F f ). Scale bar = 10 µm I) Graph shows normalized SypHy fluorescence over time in NC and Mfn2 siRNA treated with DMSO or MCUi4, each timepoint ( F ) was normalized to average pre stimulation fluorescence levels (F pre ). After one minute of basal recording, cells were depolarized with 50 mM KCl ( n = 16–19 cells from 4 independent cultures). Ratiometric difference between F f /F 0 was shown to be significant p ≤ 0.05 between Mfn2 DMSO and Mfn2 MCUi4. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Data shown as mean ± SEM.

Article Snippet: The following antibodies were used: Actin (Sigma-Aldrich, St. Louis, MO, USA, #A4700 1:2500), Actin (Sigma-Aldrich, #A5441 1:2500), LC3 (Cell Signaling Technology, Danvers, MA, USA #3868 1:1000), Mfn2 (Abcam, Cambridge, UK, #Ab56889 1:1000), Neuropeptide Y (Cell Signaling Technology, #11976T 1:500), pSer293-PDH (Merck Millipore, Burlington, MA, USA #ABS204 1:1000), PDH (Santa Cruz, Dallas, TX, USA #sc-377,092), SNAP25 (Biolegend, San Diego, CA, USA #850301 1:1000), Synapsin-1 (Cell Signaling Technology, #5297T 1:1000), Synaptophysin (Abcam,#Ab14692 1:1000), Synaptotagmin-1 (Novus Biologicals, Littleton, CO, USA #MAB4364-SP 1:500), Syntaxin-1 (Sigma-Aldrich, #S0664) 1:500), TIM23 (BD Biosciences, Franklin Lakes, NJ, USA #611223 1:1000), and VDAC1 (Abcam, #Ab14734 1:1000).

Techniques: Inhibition, Western Blot, Control, Fluorescence

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: Molecular medicine reports

Article Title: Survivin activates NF‑κB p65 via the IKKβ promoter in esophageal squamous cell carcinoma.

doi: 10.3892/mmr.2015.4737

Figure Lengend Snippet: Figure 1. Survivin knockdown results in downregulation of IKKα, IΚΚβ and NF‑κB p65 in Eca109 and KYSE150 cells. Eca109 and KYSE150 cells were transfected with LV3‑survivin shRNA plasmid (Eca109 survivin KD and KYSE150 survivin KD) and LV3‑survivin control plasmids (Eca109 control and KYSE150 control), respectively. The expression levels of survivin, NF‑κB, IΚΚα and IΚΚβ were measured by quantitative reverse transcrip tion‑polymerase chain reaction and western blotting in (A and B) Eca109 cells and (C and D) KYSE150 cells. GADPH served as an internal and loading control. Columns present the mean values from triplicate experiments and the error bars indicate the standard deviation. *P<0.05; **P<0.01 vs. control. KD, knockdown; NF‑κB p65, transcription factor p65; IKKα, inhibitor of nuclear factor κB kinase subunit α; IΚΚβ, inhibitor of nuclear factor κB kinase subunit β; p, phosphorylated.

Article Snippet: The membranes were blocked with 5% non-fat dried milk (Sigma-Aldrich) in Tris-buffered saline and Tween-20 (TBST; Beijing Solarbio Science & Technology Co., Ltd.,Beijing,China) at room temperature for 2 h. The membranes were incubated overnight at 4 ̊C with the following primary antibodies: Rabbit polyclonal anti-GAPDH (1:400; cat. no. BA2913; Wuhan Boster Biological Technology, Ltd.), which served as a loading control; rabbit polyclonal anti-survivin (1:1,000; cat. no. sc-10811; Santa Cruz Biotechnology Inc.); rabbit polyclonal anti-phosphorylated (p)-NF-κB p65 (pSer536) (1:1,000; cat. no. AF2006; Affinity Biosciences, Cell Signal Transduction, Cincinnati, OH, USA); rabbit polyclonal anti-NF-κB p65 (1:400; cat. no. BA0610; Wuhan Boster Biological Technology, Ltd.); rabbit polyclonal anti‐IΚΚα (1:400; cat. no. BA1594-2; Wuhan Boster Biological Technology, Ltd.); and rabbit polyclonal anti‐IΚΚβ (1:400; cat. no. BA4458-2; Wuhan Boster Biological Technology, Ltd.) were blocked in TBST.

Techniques: Knockdown, Transfection, shRNA, Plasmid Preparation, Control, Expressing, Western Blot, Standard Deviation

Journal: Molecular medicine reports

Article Title: Survivin activates NF‑κB p65 via the IKKβ promoter in esophageal squamous cell carcinoma.

doi: 10.3892/mmr.2015.4737

Figure Lengend Snippet: Figure 2. YM155 inhibited the expression of survivin, NF‑κB p65, IKKα and IΚΚβ in Eca109 and KYSE150 cells. (A) Eca109 and KYSE150 cells were incubated with 0.005, 0.05, 0.5, 5 and 50 µM YM155 for 48 h. Cells incubated with the culture medium served as the control group. Experiments were performed in triplicate. Eca109 and KYSE150 cells were treated with 0 (blank), 0.05, 0.5, and 5.0 µM YM155. The expression of survivin, NF‑κB p65, IΚΚβ and IΚΚα were analyzed by (B and C) western blotting and (D and E) RT‑qPCR. Western blotting of survivin, p‑NF‑κB p65, NF‑κB p65, IΚΚβ and IΚΚα proteins was conducted using total protein isolated from cells, with GADPH serving as a loading control. Transcript levels were measured by RT‑qPCR of total isolated RNA, with GADPH serving as an internal control. YM155 inhibited expression of survivin and NF‑κB p65, IKKα and IΚΚβ in (B and D) Eca109 and (C and E) KYSE150 cells. Columns demonstrate the mean values from triplicate experiments and the error bars indicate standard deviation. *P<0.05; **P<0.01 vs. control. RT‑qPCR, quantitative reverse transcription‑polymerase chain reaction; NF‑κB p65, transcription factor p65; IKKα, inhibitor of nuclear factor κB kinase subunit α; IΚΚβ, inhibitor of nuclear factor κB kinase subunit β; p, phosphorylated; mRNA, messenger RNA.

Article Snippet: The membranes were blocked with 5% non-fat dried milk (Sigma-Aldrich) in Tris-buffered saline and Tween-20 (TBST; Beijing Solarbio Science & Technology Co., Ltd.,Beijing,China) at room temperature for 2 h. The membranes were incubated overnight at 4 ̊C with the following primary antibodies: Rabbit polyclonal anti-GAPDH (1:400; cat. no. BA2913; Wuhan Boster Biological Technology, Ltd.), which served as a loading control; rabbit polyclonal anti-survivin (1:1,000; cat. no. sc-10811; Santa Cruz Biotechnology Inc.); rabbit polyclonal anti-phosphorylated (p)-NF-κB p65 (pSer536) (1:1,000; cat. no. AF2006; Affinity Biosciences, Cell Signal Transduction, Cincinnati, OH, USA); rabbit polyclonal anti-NF-κB p65 (1:400; cat. no. BA0610; Wuhan Boster Biological Technology, Ltd.); rabbit polyclonal anti‐IΚΚα (1:400; cat. no. BA1594-2; Wuhan Boster Biological Technology, Ltd.); and rabbit polyclonal anti‐IΚΚβ (1:400; cat. no. BA4458-2; Wuhan Boster Biological Technology, Ltd.) were blocked in TBST.

Techniques: Expressing, Incubation, Control, Western Blot, Isolation, Standard Deviation, Reverse Transcription Polymerase Chain Reaction

Journal: Molecular medicine reports

Article Title: Survivin activates NF‑κB p65 via the IKKβ promoter in esophageal squamous cell carcinoma.

doi: 10.3892/mmr.2015.4737

Figure Lengend Snippet: Figure 3. Survivin overexpression results in upregulation of NF‑κBp65, IKKα, IΚΚβ in Eca109 and KYSE150cells. (A and B) Eca109 and (C and D) KYSE150 cells were transfected with GV142‑survivin overexpression plasmid (Eca109 survivin OE and KYSE150 survivin OE), GV142‑control plasmid (Eca109 control and KYSE150 control), and mock transfection. Expression levels of survivin, NF‑κB p65, IΚΚβ, and IΚΚα were analyzed by (A and C) RT‑qPCR and (B and D) western blotting of total protein. Transcript levels were measured by RT‑qPCR of total isolated RNA, with GADPH serving as an internal control. Columns indicate the mean values from triplicate experiments and the error bars indicate standard deviation. *P<0.05; **P<0.01 vs. control. OE, overexpression; RT‑qPCR, quantitative reverse transcription‑polymerase chain reaction; NF‑κB p65, transcription factor p65; IKKα, inhibitor of nuclear factor κB kinase subunit α; IΚΚβ, inhibitor of nuclear factor κB kinase subunit β; p, phosphorylated; mRNA, messenger RNA.

Article Snippet: The membranes were blocked with 5% non-fat dried milk (Sigma-Aldrich) in Tris-buffered saline and Tween-20 (TBST; Beijing Solarbio Science & Technology Co., Ltd.,Beijing,China) at room temperature for 2 h. The membranes were incubated overnight at 4 ̊C with the following primary antibodies: Rabbit polyclonal anti-GAPDH (1:400; cat. no. BA2913; Wuhan Boster Biological Technology, Ltd.), which served as a loading control; rabbit polyclonal anti-survivin (1:1,000; cat. no. sc-10811; Santa Cruz Biotechnology Inc.); rabbit polyclonal anti-phosphorylated (p)-NF-κB p65 (pSer536) (1:1,000; cat. no. AF2006; Affinity Biosciences, Cell Signal Transduction, Cincinnati, OH, USA); rabbit polyclonal anti-NF-κB p65 (1:400; cat. no. BA0610; Wuhan Boster Biological Technology, Ltd.); rabbit polyclonal anti‐IΚΚα (1:400; cat. no. BA1594-2; Wuhan Boster Biological Technology, Ltd.); and rabbit polyclonal anti‐IΚΚβ (1:400; cat. no. BA4458-2; Wuhan Boster Biological Technology, Ltd.) were blocked in TBST.

Techniques: Over Expression, Transfection, Plasmid Preparation, Control, Expressing, Western Blot, Isolation, Standard Deviation, Reverse Transcription Polymerase Chain Reaction

Journal: Frontiers in Synaptic Neuroscience

Article Title: Identification of Synaptic DGKθ Interactors That Stimulate DGKθ Activity

doi: 10.3389/fnsyn.2022.855673

Figure Lengend Snippet:

Article Snippet: Rabbit anti-synaptotagmin1 , Cell Signaling Technology , 14558S , – , 1:1000.

Techniques: Purification

Journal: ACS chemical neuroscience

Article Title: Chronic Social Isolation Stress during Peri-Adolescence Alters Presynaptic Dopamine Terminal Dynamics via Augmentation in Accumbal Dopamine Availability

doi: 10.1021/acschemneuro.8b00360

Figure Lengend Snippet: Assessment of several terminal protein expression levels. Relative protein expression levels of (A) VMAT2 (n = 7 in both groups), (B) Synaptigyrin-3 (aGH: n = 6; aSI: n = 5), (C) Syntaxin-1 (aGH: n = 7; aSI: n = 6), and (D) Munc13-3 (aGH: n = 8; aSI: n = 8) were measured. None of these proteins had different expression levels in aGH and aSI rats. (insets) The representative Western blot images, with the respective protein and actin for comparison. Group housed, aGH, blue; Socially isolated, aSI, red.

Article Snippet: Subsequently, blots were incubated with agitation for 2 h at room temperature in TBS-T/5% bovine serum albumin (05470; Sigma-Aldrich) solution containing the following primary antibody concentrations: VAMT2 (1:2000; AB1598P; Millipore Sigma); Synaptogyrin-3 (1:1000; ab106460; abcam); Syntaxin-1 (1:1000; ANR-002; Alomone laboratories).

Techniques: Expressing, Western Blot, Isolation