Journal: Journal of Neuroinflammation

Article Title: Actions of the antihistaminergic clemastine on presymptomatic SOD1-G93A mice ameliorate ALS disease progression

doi: 10.1186/s12974-016-0658-8

Figure Lengend Snippet: Clemastine short treatment affects the spinal cord pathology in SOD1-G93A mice at symptomatic phase of the disease. a Schematic representation of experimental design. b Spinal cord sections (L3–L5) from WT (~120 days) and vehicle- or clemastine-treated SOD1-G93A mice at PND 120 ( n = 4/group) are stained with cresyl violet (scale bar 100 μm) and assessment of motoneuron number is performed by percentage quantification ( n = 4/group) ( t test referred to WT * p < 0.05 or to vehicle-SOD1-G93A, # p < 0.05). c Spinal cord sections of vehicle- or clemastine-treated SOD1-G93A mice at PND 120 were then stained with anti-NeuN (scale bar = 40 μm). d Equal amounts of total lumbar spinal cord lysates from WT mice (~120 days) and vehicle- or clemastine-treated SOD1-G93A mice at PND 120 (n = 3/group) are subjected to western blotting with anti-Iba1, anti-CD68, anti-ARG-1, anti-CD163 and anti-GAPDH for protein normalisation. Data represent mean ± S.E.M. Statistical significance is calculated by student’s t test referred to WT, * p < 0.05, or to vehicle SOD1-G93A mice, # p < 0.05. e Equal amounts of lumbar spinal cord lysates from vehicle- or clemastine-treated SOD1-G93A mice at PND 120 ( n = 3/group) are subjected to western blotting with anti-P2X7, anti-P2X4, anti-P2Y12 and anti-GAPDH. Data represent mean ± S.E.M. Statistical significance was calculated by student’s t test, as referred to vehicle-treated SOD1-G93A mice, * p < 0.05

Article Snippet: After saturation with 5 % non-fat dry milk, blots were probed overnight at 4 °C with the specific primary antibodies: rabbit anti-ARG1 (1:1000, Millipore, USA), rat anti-CD68 (1:500, AbD Serotech, UK), rabbit anti-CD163 (1:100, Santa Cruz Biotechnology, USA), rabbit anti-Iba1 (1:1000, Wako, USA), rabbit anti-LC3B (1:1000, Cell Signaling Technology Inc, USA), rabbit anti-mTOR and phospho-mTOR (1:1000, Cell Signaling Technology Inc), rabbit anti-nuclear factor-kappa B (NF-kB) (1:500, Cell Signaling Technology Inc), rabbit anti-P2X4 (1:500, Alomone Labs, Israel), rabbit anti-P2X7 (1:500, Alomone Labs), rabbit anti-P2Y12 (1:200, Anaspec, USA), anti-p44/42 MAPK (ERK1/2) (L34F12) mouse antibody (1:1000) and anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1:1000, Cell Signaling Technology Inc.), rabbit anti-SOD1 (1:1000, Enzo Life sciences, USA), mouse anti-SQSTM1/p62 (1:1000, Abcam, USA).

Techniques: Staining, Western Blot

Journal: Journal of Neuroinflammation

Article Title: Actions of the antihistaminergic clemastine on presymptomatic SOD1-G93A mice ameliorate ALS disease progression

doi: 10.1186/s12974-016-0658-8

Figure Lengend Snippet: Clemastine modulates the activation pattern of SOD1-G93A spinal microglia cultures from symptomatic mice. Spinal primary microglia from SOD1-G93A mice at PND 120 are exposed to clemastine 30 μM for 6 h. a Equal amounts of total lysates are subjected to western blotting with anti-CD68, anti-ARG-1, anti-CD163, anti-P2X7, anti-P2X4, anti-P2Y12 and anti-GAPDH for protein normalisation. Data represent mean ± S.E.M. of n = 3 independent experiments. Statistical significance is calculated by student’s t test referred to untreated cells ( Ctrl ), * p < 0.05. Cells are then analysed by means of a confocal microscope (scale bar 20 μm) after staining with anti-CD11b ( red ) and in b with anti-CD68 ( green ), in c with anti-ARG-1 ( green ), in d with anti-CD163 ( green ) and in e with anti-P2Y12 ( green )

Article Snippet: After saturation with 5 % non-fat dry milk, blots were probed overnight at 4 °C with the specific primary antibodies: rabbit anti-ARG1 (1:1000, Millipore, USA), rat anti-CD68 (1:500, AbD Serotech, UK), rabbit anti-CD163 (1:100, Santa Cruz Biotechnology, USA), rabbit anti-Iba1 (1:1000, Wako, USA), rabbit anti-LC3B (1:1000, Cell Signaling Technology Inc, USA), rabbit anti-mTOR and phospho-mTOR (1:1000, Cell Signaling Technology Inc), rabbit anti-nuclear factor-kappa B (NF-kB) (1:500, Cell Signaling Technology Inc), rabbit anti-P2X4 (1:500, Alomone Labs, Israel), rabbit anti-P2X7 (1:500, Alomone Labs), rabbit anti-P2Y12 (1:200, Anaspec, USA), anti-p44/42 MAPK (ERK1/2) (L34F12) mouse antibody (1:1000) and anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1:1000, Cell Signaling Technology Inc.), rabbit anti-SOD1 (1:1000, Enzo Life sciences, USA), mouse anti-SQSTM1/p62 (1:1000, Abcam, USA).

Techniques: Activation Assay, Western Blot, Microscopy, Staining

Journal: BMC Neuroscience

Article Title: Functional crosstalk in culture between macrophages and trigeminal sensory neurons of a mouse genetic model of migraine

doi: 10.1186/1471-2202-13-143

Figure Lengend Snippet: Activated phenotype of trigeminal ganglion macrophages in vitro. A , Examples of fluorescence microscopy images of cultures from WT or KI trigeminal ganglia immunostained with antibodies against Iba1 (red), β˜-tubulin III (green) and counterstained with DAPI (blue). MФ. indicates co-culturing with known number of host macrophages, while only MФ .indicates pure peritoneal macrophage cultures. Scale bar: 50 μm. B , Histograms quantify the number of Iba1-positive cells in the ROI (640 × 480 μm) from WT or R129Q KI ganglion cultures in control condition and after MФ. addition; n = 3 petri dishes for each group run in parallel, * p < 0.05, ** p < 0.01. MФ .# p < 0.01 vs all conditions. C , Histograms quantify the average cell area (expressed in μm 2 ) for data shown in B , * p < 0.05, ** p < 0.01. D , P2X3 mRNA levels in co-cultures (WT+MФ, KI+MФ) expressed as fold increase in comparison to their controls (WT or R192Q KI cultures) and normalized versus β-tubulin mRNA levels (n = 3 petri dishes for each group). E , Example of western blots shows similar levels of P2X3 receptor expression in lysates from WT or R192Q KI trigeminal cultures (lanes 1 and 3), and in co-cultures (WT+MФ and KI+MФ, lanes 2 and 4). Lysates from MФ only (lane 5) show no signal. β-Tubulin III and actin are shown as loading control (bottom row). F , Histograms show mean values (optical density, AUs) of P2X3 subunits obtained in western blot experiments normalized over β-tubulin III signals; n = 4 petri dishes.

Article Snippet: Western blotting was performed as described earlier [ , ], using antibodies against anti-P2X3 (1:300; Alomone, Jerusalem, Israel), anti-β-tubulin III (1:2.000; Sigma) or anti-actin (1:3.000; Sigma).

Techniques: In Vitro, Fluorescence, Microscopy, Control, Comparison, Western Blot, Expressing

Journal: BMC Neuroscience

Article Title: Functional crosstalk in culture between macrophages and trigeminal sensory neurons of a mouse genetic model of migraine

doi: 10.1186/1471-2202-13-143

Figure Lengend Snippet: Neuronal P2X3 receptor-mediated responses in basal conditions or in the presence of host macrophages. A , Representative examples of currents induced by application of α,β-meATP (10 μM, 2 s; horizontal bar) to trigeminal neurons from WT ( n = 15 neurons) or R192Q KI ( n = 17) cultures in standard conditions (left traces) or when co-cultured with macrophages (WT+MФ, . n = 24; KI+MФ, . n = 20. Note that macrophage co-culturing increases P2X3-mediated responses from WT neurons. Average data are plotted in B . * p < 0.05; ** p < 0.01. C, Rise time (left; expressed as time from 10 to 90% of peak amplitude), desensitization onset (middle; expressed as the first time constant, τ fast , of current decay). Values are from 13-23 neurons. Recovery from desensitization (right; expressed as % of control amplitude in a paired pulse agonist application) was faster for WT+MФ vs WT; * p = 0.007. All responses were evoked by α,β-meATP (10 μM, 2 s). D , ATP medium content measured 5 h in WT or KI trigeminal neuron-macrophage co-cultures. Basal ATP levels present in culture macrophages are also shown. Data are expressed as fractional increase with respect to neuronal WT or KI cultures. n = 2, * p < 0.05.

Article Snippet: Western blotting was performed as described earlier [ , ], using antibodies against anti-P2X3 (1:300; Alomone, Jerusalem, Israel), anti-β-tubulin III (1:2.000; Sigma) or anti-actin (1:3.000; Sigma).

Techniques: Cell Culture, Control

Journal: Antioxidants & Redox Signaling

Article Title: Oxidative Stress in HIV Infection and Alcohol Use: Role of Redox Signals in Modulation of Lipid Rafts and ATP-Binding Cassette Transporters

doi: 10.1089/ars.2016.6830

Figure Lengend Snippet: Effect of ABC transporters gene expression. PBMCs (3 × 106 cells/ml) were treated with alcohol (0.1%), HIV-1 gp120 (50 ng/ml), and AL with HIV-1 gp120 at 24 h. RNA was extracted and reverse transcribed followed by quantitative real-time PCR for ABCA1 (A), ABCC1 (B), ABCG1 (C), ABCG4 (D), and housekeeping β-actin-specific primers. Data are expressed as mean ± SE of TAI values of three independent experiments. ABC, ATP-binding cassette.

Article Snippet: After blocking, the membrane was probed with primary rabbit polyclonal GSS, GPx (One world lab, CA), SREBP-2, (Cayman, CA) Cav-1, HMGCR (Santa Cruz Biotechnology, CA), DHCR-7 (GeneTex, Inc., Irvine, CA), FABP (Abnova, Taiwan), ABC transporters ABCA1, ABCC1, ABCG1, and ABCC4 (Boster Biological Technology, Pleasanton, CA), and mouse monoclonal SOD antibody (Chemicon International, CA); this was followed by secondary goat antirabbit IgG antibody and goat antimouse IgG antibody (Santa Cruz Biotechnology, CA).

Techniques: Gene Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Binding Assay

Journal: Antioxidants & Redox Signaling

Article Title: Oxidative Stress in HIV Infection and Alcohol Use: Role of Redox Signals in Modulation of Lipid Rafts and ATP-Binding Cassette Transporters

doi: 10.1089/ars.2016.6830

Figure Lengend Snippet: HIV-1 gp120 with alcohol effect of ABC transporters. PBMCs (3 × 106 cells/ml) were treated with HIV-1 gp120 (50 ng), alcohol (0.1%), and a combination of HIV-1 gp120 with AL. Controls were maintained by drug-free medium. At the end of the incubation, equal amounts of protein lysate were resolved by 4%–15% SDS-PAGE and protein expression was analyzed by Western blot showing ABCA1 (A), ABCC1 (B), ABCG1 (C), and ABCG4 (D). Data presented in (E–H) show the densitometry evaluation expressed as mean ± SE of minimum three independent experiments.

Article Snippet: After blocking, the membrane was probed with primary rabbit polyclonal GSS, GPx (One world lab, CA), SREBP-2, (Cayman, CA) Cav-1, HMGCR (Santa Cruz Biotechnology, CA), DHCR-7 (GeneTex, Inc., Irvine, CA), FABP (Abnova, Taiwan), ABC transporters ABCA1, ABCC1, ABCG1, and ABCC4 (Boster Biological Technology, Pleasanton, CA), and mouse monoclonal SOD antibody (Chemicon International, CA); this was followed by secondary goat antirabbit IgG antibody and goat antimouse IgG antibody (Santa Cruz Biotechnology, CA).

Techniques: Incubation, SDS Page, Expressing, Western Blot

Journal: Purinergic Signalling

Article Title: Distribution of purinergic P2X receptors in the equine digit, cervical spinal cord and dorsal root ganglia

doi: 10.1007/s11302-013-9356-5

Figure Lengend Snippet: Primer sequences (forward/reverse) for equine P2X 1−7 cDNAs

Article Snippet: Membranes were blocked in 5 % skimmed dry milk in phosphate-buffered saline for 1 h at room temperature and incubated overnight at 4 °C with the primary antibody in 0.1 % Tween blocking solution, (1) anti-P2X rabbit polyclonal antibody (Alomone) 1:200, (2) anti-P2X 2 rabbit polyclonal antibody (Abcam) 0.6 μg/mL, (3) anti-P2X 3 rabbit polyclonal antibody (Neuromics) 1:1,000, (4) anti-P2X 4 whole serum rabbit antibody (Abcam) 1:300, (5) anti-P2X 5 rabbit polyclonal antibody (Alomone) 1:200, (6) anti-P2X 6 rabbit polyclonal antibody (Abcam) 10 μg/mL or (7) anti-P2X 7 rabbit polyclonal antibody (Alomone) 1:200.

Techniques: Sequencing, Amplification

Journal: Purinergic Signalling

Article Title: Distribution of purinergic P2X receptors in the equine digit, cervical spinal cord and dorsal root ganglia

doi: 10.1007/s11302-013-9356-5

Figure Lengend Snippet: Summary of Western blot analysis plus predicted sizes for P2X 1–3, 7 proteins

Article Snippet: Membranes were blocked in 5 % skimmed dry milk in phosphate-buffered saline for 1 h at room temperature and incubated overnight at 4 °C with the primary antibody in 0.1 % Tween blocking solution, (1) anti-P2X rabbit polyclonal antibody (Alomone) 1:200, (2) anti-P2X 2 rabbit polyclonal antibody (Abcam) 0.6 μg/mL, (3) anti-P2X 3 rabbit polyclonal antibody (Neuromics) 1:1,000, (4) anti-P2X 4 whole serum rabbit antibody (Abcam) 1:300, (5) anti-P2X 5 rabbit polyclonal antibody (Alomone) 1:200, (6) anti-P2X 6 rabbit polyclonal antibody (Abcam) 10 μg/mL or (7) anti-P2X 7 rabbit polyclonal antibody (Alomone) 1:200.

Techniques: Western Blot, Molecular Weight

Journal: Signal Transduction and Targeted Therapy

Article Title: Nucleoside reverse transcriptase inhibitors and Kamuvudines inhibit amyloid-β induced retinal pigmented epithelium degeneration

doi: 10.1038/s41392-021-00537-z

Figure Lengend Snippet: P2RX7 expression is required for AβOs-induced RPE degeneration. Eyes were treated with a single subretinal injection of 1 μM AβOs. Tissue was collected 7 days after injection. a P2rx7 −/− mice are protected from AβOs-induced RPE degeneration, n = 8. b Lower magnification (left panel) and higher magnification (right panel) of RPE flat mounts of P2rx7 hP2RX7Flox and P2rx7 hP2RX7Flox / Best1-Cre+ mice stained with phalloidin (white) and P2RX7 (yellow) demonstrating reduction of P2RX7 signal in the RPE of P2rx7 hP2RX7Flox / Best1-Cre+ mice compared to P2rx7 hP2RX7Flox mice. Black arrowhead points to the optic nerve of P2rx7 hP2RX7Flox / Best1-Cre+ mice, where expression of P2RX persists in non-RPE tissue. c AβOs induced degeneration in P2rx7 hP2RX7Flox ( n = 6) but not in P2rx7 hP2RX7Flox / Best1-Cre+ mice ( n = 8) ( d ). Representative images are shown. Fundus photographs, top row; Flat mounts stained for zonula occludens-1 (ZO-1; red), bottom row. Degeneration outlined by white arrowheads. Binary (Healthy %) and morphometric (PM, polymegethism (mean (SEM)) quantification of RPE degeneration is shown (Fisher’s exact test for binary; two-tailed t -test for morphometry; * P < 0.01, **P < 0.001). Loss of regular hexagonal cellular boundaries in ZO-1 stained flat mounts is indicative of degenerated RPE. Scale bars for lower magnification (100 μm) and higher magnification (50 μm)

Article Snippet: The RPE flat mounts were stained with Dylight phalloidin 650 (1:10, Cell Signaling) and a rabbit polyclonal anti-P2RX7 (extracellular) antibody (1:100, Alomone Labs), followed by a goat anti-rabbit Alexa-555 antibody (1:200, Invitrogen).

Techniques: Expressing, Injection, Staining, Two Tailed Test