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