Journal: Cellular and Molecular Neurobiology

Article Title: Neurotrophic Activity of Cultured Cell Line U87 is Up-Regulated by Proline-Rich Polypeptide Complex and Its Constituent Nonapeptide

doi: 10.1007/s10571-015-0192-8

Figure Lengend Snippet: Western blotting analysis of NGF forms secreted by astrocytoma cell line U87. Supernatants from control and compound-treated cells were separated in SDS-PAGE, then transferred to nitrocellulose membrane. NGF forms were detected with the use of specific polyclonal antibodies anti-βNGF. Immunocomplexes were visualized using a NBT/BCIP substrate and analyzed densitometrically in Molecular Imager ChemiDoc MP Imaging System with Image Lab Softwere (BioRad). The representative immunoblot is presented

Article Snippet: Rabbit anti-BDNF polyclonal antibody was from Bioss (MA, USA).

Techniques: Western Blot, SDS Page, Imaging

Journal: Journal of Alzheimer's Disease

Article Title: Intraneuronal Amylin Deposition, Peroxidative Membrane Injury and Increased IL-1β Synthesis in Brains of Alzheimer’s Disease Patients with Type-2 Diabetes and in Diabetic HIP Rats

doi: 10.3233/jad-160047

Figure Lengend Snippet: Fig. 1. Testing the amylin deposition in human brain specimens by HPLC-MS/MS. A) Typical reverse phase HPLC chromatogram of human amylin in human amylin standard and human brain lysates. B) Fractions of human brain lysates collected by HPLC were immunoblotted with a rabbit anti-amylin antibody. 10 ng of recombinant human amylin (H-Amy) was loaded as positive control. A representative blot from 3 independent experiments is shown. C) Fractions collected from HPLC (the amylin positive fractions) were analyzed by LC-MS/MS. The representative mass spectrum of peptides eluted at 27.54 min shows the 4+ ion of amylin (m/z = 976.23). D) The tandem MS/MS spectrum of amylin. The fragments corresponding to the b ions (amylin ions) are labeled proving that this peptide is amylin. E) Tandem MS/MS spectra of amylin in three additional brain samples from T2D-AD group. The amylin peak m/z = 976.23200 ± 10 ppm is extracted and intensity is shown in each spectrum.

Article Snippet: For testing the presence of amylin human brain specimens, the amylin-containing tissue lysate fractions were analyzed by LC-MS/MS using an LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA) coupled with an Eksigent Nanoflex cHiPLCTM system (Eksigent, Dublin, CA) through a nano-electrospray ionization source [25].

Techniques: Tandem Mass Spectroscopy, Recombinant, Positive Control, Liquid Chromatography with Mass Spectroscopy, Labeling

Journal: Journal of Alzheimer's Disease

Article Title: Intraneuronal Amylin Deposition, Peroxidative Membrane Injury and Increased IL-1β Synthesis in Brains of Alzheimer’s Disease Patients with Type-2 Diabetes and in Diabetic HIP Rats

doi: 10.3233/jad-160047

Figure Lengend Snippet: Fig. 2. Amylin deposition in brain specimens from the T2D-AD patient group demonstrated by immunohistochemistry with an anti-amylin antibody on thin tissue sections. A) Representative image of amylin deposition in brain blood vessels and parenchyma. B–D) Neurons showing immunoreactivity for amylin. Neuron amylin immunoreactivity may result from amylin deposition around neurons, as suggested by images in B and D (arrows). Some neurons (D, arrows) appear to accumulate cytosolic amylin immunoreactive inclusions. Red arrows in C point to A deposits (red) which form mixed plaques with amylin (brown), as demonstrated by prior data [9, 11]. Brain sections of non-diabetic cognitively normal individuals lack cytosolic amylin immunoreactive inclusions (E). Scale bars, 200 m in A and 20 m in B–E.

Article Snippet: For testing the presence of amylin human brain specimens, the amylin-containing tissue lysate fractions were analyzed by LC-MS/MS using an LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA) coupled with an Eksigent Nanoflex cHiPLCTM system (Eksigent, Dublin, CA) through a nano-electrospray ionization source [25].

Techniques: Immunohistochemistry

Journal: Journal of Alzheimer's Disease

Article Title: Intraneuronal Amylin Deposition, Peroxidative Membrane Injury and Increased IL-1β Synthesis in Brains of Alzheimer’s Disease Patients with Type-2 Diabetes and in Diabetic HIP Rats

doi: 10.3233/jad-160047

Figure Lengend Snippet: Fig. 3. Incorporation of aggregated amylin in brains of humans is associated with peroxidative membrane injury. A–C) Dual immunofluo- rescence staining of amylin (green) and 4-HNE (MDA) (red) in the brain (A and B) and pancreas (C) of patients with T2D and non-diabetic controls (Ctl). Nuclei are shown in blue in the overlay images. Bar graphs display the mean pixel-by-pixel covariance in amylin and 4- HNE(MDA) images (Pearson’s correlation coefficient; PCC) in brains from patients with T2D versus Ctl. Scale bars, 20 m. 10 sections per sample from n = 4 individuals in each group were investigated. In (D), 4-HNE and MDA were immunoprecipitated from brain homogenates. The level of amylin in the fractions enriched in 4-HNE and MDA was then quantified by ELISA. Data are presented as mean ± standard error. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: For testing the presence of amylin human brain specimens, the amylin-containing tissue lysate fractions were analyzed by LC-MS/MS using an LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA) coupled with an Eksigent Nanoflex cHiPLCTM system (Eksigent, Dublin, CA) through a nano-electrospray ionization source [25].

Techniques: Membrane, Staining, Immunoprecipitation, Enzyme-linked Immunosorbent Assay

Journal: Journal of Alzheimer's Disease

Article Title: Intraneuronal Amylin Deposition, Peroxidative Membrane Injury and Increased IL-1β Synthesis in Brains of Alzheimer’s Disease Patients with Type-2 Diabetes and in Diabetic HIP Rats

doi: 10.3233/jad-160047

Figure Lengend Snippet: Fig. 4. Incorporation of aggregated amylin in the brains of humans is associated IL-1 accumulation. Immunofluorescence staining of amylin (green) and IL-1 (red) in the brain specimens from diabetic humans. The bar graph shows the number of neurons that are positive for both amylin and IL-1 in a 134 × 134 m area in brain sections from patients in the diabetic group versus controls. Scale bars, 20 m. 10 sections per sample from n = 4 individuals in each group were investigated. Data are presented as mean ± standard error. ∗∗∗p < 0.001.

Article Snippet: For testing the presence of amylin human brain specimens, the amylin-containing tissue lysate fractions were analyzed by LC-MS/MS using an LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA) coupled with an Eksigent Nanoflex cHiPLCTM system (Eksigent, Dublin, CA) through a nano-electrospray ionization source [25].

Techniques: Staining

Journal: Journal of Alzheimer's Disease

Article Title: Intraneuronal Amylin Deposition, Peroxidative Membrane Injury and Increased IL-1β Synthesis in Brains of Alzheimer’s Disease Patients with Type-2 Diabetes and in Diabetic HIP Rats

doi: 10.3233/jad-160047

Figure Lengend Snippet: Fig. 5. Aggregated amylin induces peroxidative membrane injury in brain in diabetic rats expressing human amylin in the pancreas. A) A LC-MS/MS method was applied to measure MDA in brain homogenates using derivatization of MDA with 2,4-dinitrophenylhydrazine (DNPH). Same method was applied to measure GSH-HNE in rat brain homogenates. B) Dual-immunofluorescence staining of amylin (green) and 4-HNE (or MDA; red) in brain tissue sections from HIP rats. Bar graphs display the mean pixel-by-pixel covariance in amylin and 4-HNE(MDA) images (PCC) in brains from HIP versus WT rats. C) Same as in above for UCD rats (negative controls). D) Proximity ligation assay (PLA) was performed with anti-amylin and anti-4-HNE antibodies on brain sections. PLA signal shows an overall consistency with 4-HNE-amylin appearing in clusters. Nuclei are shown in blue in the overlay image. 10 sections per sample from n = 4 rats in each group were investigated. Scale bars, 20 m. E) 4-HNE or MDA was immunoprecipitated from brain homogenates obtained from HIP and WT rats and the level of amylin in fractions enriched in 4-HNE/MDA was quantified by western blot with an amylin antibody. A representative blot from 2 independent experiments is shown. Data are presented as mean ± standard error. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: For testing the presence of amylin human brain specimens, the amylin-containing tissue lysate fractions were analyzed by LC-MS/MS using an LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA) coupled with an Eksigent Nanoflex cHiPLCTM system (Eksigent, Dublin, CA) through a nano-electrospray ionization source [25].

Techniques: Membrane, Expressing, Liquid Chromatography with Mass Spectroscopy, Staining, Proximity Ligation Assay, Immunoprecipitation, Western Blot

Journal: Journal of Alzheimer's Disease

Article Title: Intraneuronal Amylin Deposition, Peroxidative Membrane Injury and Increased IL-1β Synthesis in Brains of Alzheimer’s Disease Patients with Type-2 Diabetes and in Diabetic HIP Rats

doi: 10.3233/jad-160047

Figure Lengend Snippet: Fig. 6. Aggregated amylin increases IL-1 levesl in the brains of rats expressing human amylin in the pancreas. A) Brain tissues from HIP rats were analyzed by immunofluorescence imaging with amylin and IL-1 antibodies. Neurons staining for both amylin (green) and IL-1 (red) are readily observed. The bar graph shows the number of neurons that are positive for both amylin and IL-1 in a 134 × 134 m area in brain sections from HIP versus WT rats. B) Same as in above for UCD rats (negative controls). Scale bars, 20 m. C) Relative IL-1 protein level in whole brain homogenates from age-matched WT, UCD and HIP rats assessed by western blot. D) Relative IL-1 mRNA level in whole brain homogenates from HIP versus UCD rats measured by qRT-PCR. n = 7 samples/group. A representative blot from 2 independent experiments is shown. Data are presented as mean ± standard error. ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: For testing the presence of amylin human brain specimens, the amylin-containing tissue lysate fractions were analyzed by LC-MS/MS using an LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA) coupled with an Eksigent Nanoflex cHiPLCTM system (Eksigent, Dublin, CA) through a nano-electrospray ionization source [25].

Techniques: Expressing, Imaging, Staining, Western Blot, Quantitative RT-PCR

Journal: Journal of Alzheimer's Disease

Article Title: Intraneuronal Amylin Deposition, Peroxidative Membrane Injury and Increased IL-1β Synthesis in Brains of Alzheimer’s Disease Patients with Type-2 Diabetes and in Diabetic HIP Rats

doi: 10.3233/jad-160047

Figure Lengend Snippet: Fig. 7. Intravenous injections of mice with aggregated amylin leads to formation of amylin-4-HNE/MDA adducts and IL-1 accumulation in the brain. C57BL/6 mice were intravenously injected with 2 g/g body weight aggregated human amylin (n = 4; Inj) or saline (n = 4; Ctl). A) The level of amylin in fractions from brains that were enriched in 4-HNE/MDA by immunoprecipitation was quantified by western blot. B) IL-1 levels in brain lysates from mice injected with human amylin versus control. A representative blot from 2 independent experiments is shown. Data are presented as mean ± standard error. ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: For testing the presence of amylin human brain specimens, the amylin-containing tissue lysate fractions were analyzed by LC-MS/MS using an LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA) coupled with an Eksigent Nanoflex cHiPLCTM system (Eksigent, Dublin, CA) through a nano-electrospray ionization source [25].

Techniques: Injection, Saline, Immunoprecipitation, Western Blot, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Transmission Disrupted: Modeling Auditory Synaptopathy in Zebrafish

doi: 10.3389/fcell.2018.00114

Figure Lengend Snippet: Commercially available antibodies labeling zebrafish hair-cell synaptic proteins.

Article Snippet: MAGUK , Postsynaptic densities , Mouse , Monoclonal IgG1 , 1:500 , NeuroMab (UC Davis)/ 75-029 , ; .

Techniques: Labeling, Plasmid Preparation