Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: The Effects of Brazilian Green Propolis against Excessive Light-Induced Cell Damage in Retina and Fibroblast Cells

doi: 10.1155/2013/238279

Figure Lengend Snippet: Effects of propolis and its constituents on cell damage or phosphorylated p38 induced by UVA irradiation in a 661W culture. ((a)–(e)) Cell viability was assessed by immersing cells in WST-8 solution for 6 h at 37°C, with absorbance recorded at 492 nm. UVA induced a decrease in cell viability. (a) Propolis at 10 and 30 μ g/mL significantly inhibited UVA-induced cell damage in a 661W culture. (b) 3,5-di- O -caffeoylquinic acid, (c) 3,4-di- O -caffeoylquinic acid, and (d) chlorogenic acid at 1 and 3 μ g/mL significantly inhibited cell damage, respectively. (e) p-Coumaric acid at 1 μ g/mL inhibited cell damage. (f) Representative band images showing activation of p38 in the nontreated, UVA exposure plus vehicle-treated, and UVA exposure plus propolis-treated cells. UVA exposure plus vehicle-treated group had 2 lanes. (g) Quantitative analysis of the band density of p38. Data are shown as means ± S.E.M. ( n = 6). * P < 0.05, ** P < 0.01 versus UVA exposure plus the vehicle-treated group, and ## P < 0.01 versus control. CGA: chlorogenic acid, p -CA: p -coumaric acid, 3,5-CQA: 3,5-di- O -caffeoylquinic acid, and 3,4-CQA: 3,4-di- O -caffeoylquinic acid.

Article Snippet: The membranes were incubated with the following primary antibodies: phosphorylated p38 mouse monoclonal antibody (Promega, Madison, WI, USA) (1 : 1000), phosphorylated ERK rabbit polyclonal antibody (Cell Signaling Technology Inc., Danvers, MA, USA) (1 : 1000), p38 mouse monoclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) (1 : 1000), ERK rabbit polyclonal antibody (Cell Signaling) (1 : 1000), and β -actin mouse monoclonal antibody (Sigma-Aldrich) (1 : 4000).

Techniques: Irradiation, Activation Assay, Control

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: The Effects of Brazilian Green Propolis against Excessive Light-Induced Cell Damage in Retina and Fibroblast Cells

doi: 10.1155/2013/238279

Figure Lengend Snippet: Effects of propolis on UVA-induced expression of phosphorylated p38 and ERK1/2 and MAPK inhibitors in an NB1-RGB culture. Representative Western blots (a) showing activation of p38 and ERK in the nontreated, UVA 10 J/cm 2 exposure plus vehicle-treated, and UVA exposure plus propolis-treated (30 μ g/mL) groups. Phosphorylation of p38 (b) and ERK1/2 (c) was determined by immunoblotting assay. Quantitative analysis of the band density of p38 (b) and ERK (c) by densitometric analysis. These intensities were normalized with total p38 and total ERK, respectively. (d) Cell viability was assessed by immersing cells in WST-8 solution for 6 h at 37°C, with absorbance recorded at 492 nm. SB203580 and U0126 inhibited the decrease of cellular viability induced by UVA, and the level was as high as of a propolis-treated group. Data are shown as means ± S.E.M. ( n = 6). ** P < 0.01 versus UVA exposure plus vehicle-treated cells and ## P < 0.01 versus control.

Article Snippet: The membranes were incubated with the following primary antibodies: phosphorylated p38 mouse monoclonal antibody (Promega, Madison, WI, USA) (1 : 1000), phosphorylated ERK rabbit polyclonal antibody (Cell Signaling Technology Inc., Danvers, MA, USA) (1 : 1000), p38 mouse monoclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) (1 : 1000), ERK rabbit polyclonal antibody (Cell Signaling) (1 : 1000), and β -actin mouse monoclonal antibody (Sigma-Aldrich) (1 : 4000).

Techniques: Expressing, Western Blot, Activation Assay, Phospho-proteomics, Control

Journal: Nature Communications

Article Title: Overexpression of endophilin A1 exacerbates synaptic alterations in a mouse model of Alzheimer’s disease

doi: 10.1038/s41467-018-04389-0

Figure Lengend Snippet: Effect of EP overexpression on p38 MAP kinase activation and mitochondrial dysfunction in Aβ-insulted brain in vivo and brain slices in vitro. a Brain slices from 3-month-old non-Tg or Tg Sh3gl2 mice were perfused with Aβ (50 nM) or vehicle for 1 h and then subjected to immunoblotting analysis for the phosphorylation of p38 MAP kinase (p-p38), total p38 MAP kinase (t-p38), tubulin, and β-actin. Tubulin and β-actin served as a neuronal marker and protein loading controls, respectively. Data are expressed as fold change relative to the non-Tg vehicle control group. b Immunoblotting of cortical homogenates from the indicated Tg mice at 5–6 months of age for the indicated proteins. Data are expressed as fold change relative to the non-Tg mice group. c Brain slices from indicated Tg Sh3gl2 mice were treated with vehicle or Aβ (50 nM) with/without pretreatment of EUK-134 (EUK, 500 nM), SB203580 (SB, 1 µM), or mitochondrial antioxidant MitoTEMPO (TEMPO, 1 µM) for 5 min, and then subjected to immunoblotting for the phosphorylation of p38 MAP kinase (p-P38), total p38 MAP kinase (t-p38), tubulin, and β-actin. Data are expressed as fold change relative to the Tg Sh3gl2 vehicle control group. Date are shown as mean ± s.e.m., n = 3 per group (one-way ANOVA in a – c ). d Representative spectra of EPR in non-Tg and Tg Sh3gl2 brain slices with the treatment of vehicle or Aβ (50 nM) in the presence of SB203580 (1 µM). e Quantification of EPR spectra in the indicated groups of mice. f – g Mitochondrial complex IV activity ( f ) and ATP levels ( g ) in the indicated groups of brain slices treated with vehicle or Aβ in the presence/absence of SB203580. Data are expressed as fold increase relative to non-Tg vehicle control group. Date are shown as mean ± s.e.m., n = 3 per group (one-way ANOVA in e – g )

Article Snippet: The primary antibodies used were as follows: anti-EP (Cat# 36-3000, Invitrogen), anti-phos-p38 (Cat# 612288, BD), anti-total-p38 (Cat# 9212, Cell signaling), anti-human Aβ 1-17 clone 6E10 (Cat# 9320-02, Signet), anti-synaptophysin (Cat# MAB5258; Chemicon), anti-synaptojanin 1 antibody (AC1), (Cat# MA3-936; Thermo Fisher), anti-NMDAR2B (Cat# ab81271, Abcam), anti-BACE1 antibody (Cat# ab108394, Abcam), and β-actin (Cat# A5441; Sigma-Aldrich).

Techniques: Over Expression, Activation Assay, In Vivo, In Vitro, Western Blot, Marker, Activity Assay

Journal: Nature Communications

Article Title: Overexpression of endophilin A1 exacerbates synaptic alterations in a mouse model of Alzheimer’s disease

doi: 10.1038/s41467-018-04389-0

Figure Lengend Snippet: Effect of mitochondrial ROS scavenger on EP/Aβ-mediated p38 activation, ROS production, and mitochondrial dysfunction. a Brain slices from 3-month-old non-Tg or Tg Sh3gl2 mice were perfused with Aβ (50 nM) for 1 h with/without pretreatment of mitochondrial antioxidant MitoTEMPO (TEMPO, 1 µM) for 5 min, and then subjected to measure EPR, mitochondrial complex IV activity, and ATP levels. a Representative spectrum of EPR in non-Tg and Tg Sh3gl2 brain slices with the treatment of vehicle or Aβ in the presence of MitoTEMPO. b Quantification of EPR spectra in the indicated groups of mice. c , d Mitochondrial complex IV activity ( c ) and ATP levels ( d ) in the indicated groups of brain slices treated with vehicle or Aβ in the presence/absence of MitoTEMPO. Data are expressed as fold change relative to non-Tg vehicle control group. Date are shown as mean ± s.e.m., n = 3 per group (one-way ANOVA in b – d )

Article Snippet: The primary antibodies used were as follows: anti-EP (Cat# 36-3000, Invitrogen), anti-phos-p38 (Cat# 612288, BD), anti-total-p38 (Cat# 9212, Cell signaling), anti-human Aβ 1-17 clone 6E10 (Cat# 9320-02, Signet), anti-synaptophysin (Cat# MAB5258; Chemicon), anti-synaptojanin 1 antibody (AC1), (Cat# MA3-936; Thermo Fisher), anti-NMDAR2B (Cat# ab81271, Abcam), anti-BACE1 antibody (Cat# ab108394, Abcam), and β-actin (Cat# A5441; Sigma-Aldrich).

Techniques: Activation Assay, Activity Assay

Journal: Nature Communications

Article Title: Overexpression of endophilin A1 exacerbates synaptic alterations in a mouse model of Alzheimer’s disease

doi: 10.1038/s41467-018-04389-0

Figure Lengend Snippet: Blocking EP-mediated oxidative stress and p38 activation rescued Aβ-induced synaptic loss. a , b Brain slices from 3-month-old non-Tg or Tg Sh3gl2 mice were perfused with Aβ (50 nM) for 2 h, and then subjected to immunoblotting analysis for synaptojanin ( a ) and synaptophysin ( b ) in the indicated groups of brain slices. β-Actin served as protein loading controls. The upper panel displays quantification of immunoreactive bands for the corresponding protein relative to β-actin. Data are expressed as fold change relative to the non-Tg vehicle control group. Data are shown as mean ± s.e.m., n = 3 per group (one-way ANOVA in a , b ). c , d The Tg Sh3gl2 brain slices from 3-month-old mice were perfused with Aβ (50 nM) for 2 h with/without pretreatment of 500 nM EUK-134 (EUK), 1 μM SB203580 (SB), or 1 µM MitoTEMPO (TEMPO) for 5 min. Immunoblotting for synaptojanin ( c ) and synaptophysin ( d ) in the indicated groups of brain slices. The upper panel displays the quantification of immunoreactive bands for the corresponding protein relative to β-actin. Data are expressed as fold change relative to Tg Sh3gl2 vehicle control group. Data are shown as mean ± s.e.m., n = 3 per group (one-way ANOVA in c , d ). Fourteen-day in vitro cultured cortical neurons, either non-Tg or Tg Sh3gl2 , were treated with 50 nM Aβ for 24 h, with or without 500 nM EUK-134, 1 μM SB203580, or 1 μM MitoTEMPO pretreatment for 1 h before the addition of Aβ. The numbers of synaptophysin-positive clusters were significantly decreased in Aβ-treated Tg Sh3gl2 neurons compared to vehicle-treated non-Tg neurons in e – h . Treatment with EUK-134, or SB203580, or MitoTEMPO, inhibited Aβ-induced synaptic loss in cultured EP overexpression neurons ( g , h ). Representative images for synaptophysin (green), MAP2 (red), and nuclei (blue) in the indicated groups of neurons are shown in e , g . Scale bars, 50 μm. Quantifications of synaptophysin-positive clusters per 10 μm of dendrites are shown in f , h . Data are shown as mean ± s.e.m., n = 12 cells for each group (one-way ANOVA in f , h )

Article Snippet: The primary antibodies used were as follows: anti-EP (Cat# 36-3000, Invitrogen), anti-phos-p38 (Cat# 612288, BD), anti-total-p38 (Cat# 9212, Cell signaling), anti-human Aβ 1-17 clone 6E10 (Cat# 9320-02, Signet), anti-synaptophysin (Cat# MAB5258; Chemicon), anti-synaptojanin 1 antibody (AC1), (Cat# MA3-936; Thermo Fisher), anti-NMDAR2B (Cat# ab81271, Abcam), anti-BACE1 antibody (Cat# ab108394, Abcam), and β-actin (Cat# A5441; Sigma-Aldrich).

Techniques: Blocking Assay, Activation Assay, Western Blot, In Vitro, Cell Culture, Over Expression

Journal: Nature Communications

Article Title: Overexpression of endophilin A1 exacerbates synaptic alterations in a mouse model of Alzheimer’s disease

doi: 10.1038/s41467-018-04389-0

Figure Lengend Snippet: Inhibition of p38 MAP kinase rescues impairment on synaptic plasticity and spatial learning and memory in Tg Sh3gl2 /mAPP mice. a , b Hippocampal slices from 5-month-old to 6-month-old Tg Sh3gl2 mice were pretreated with SB203580 (SB, 1 µM) for 5 min before Aβ perfusion (100 nM for 20 min) and then hippocampal CA3-CA1 LTP was recorded ( a ). Tg Sh3gl2 /mAPP mice were intraperitoneally injected with SB203580 (0.5 mg/kg) once a day for 3 weeks and then performed LTP experiments ( b ) and Morris water maze test ( c – f ) at the age of 5–5.5 months. Upper panels of a and b show representative traces of fEPSP in the indicated slices with the indicated treatment before θ-burst stimulation (black line) after 1 h (gray line). Administration of SB203850 significantly ameliorated hippocampal LTP deficit in Tg Sh3gl2 /mAPP mice compared to the vehicle-treated group. Error bars represent s.e.m., n = 7–10 per group. * p < 0.01 (one-way ANOVA in a , b ). c Escape latencies in hidden platform during Morris water maze task training in indicated groups. Error bars represent s.e.m., n = 8–9 mice per group (one-way ANOVA). d Time spent in the quadrant with the hidden platform and e mean number of crossings of the target during .the probe test. f Representative searching traces during the probe test. Data are shown as mean ± s.e.m., n = 8–9 mice per group (one-way ANOVA in d , e )

Article Snippet: The primary antibodies used were as follows: anti-EP (Cat# 36-3000, Invitrogen), anti-phos-p38 (Cat# 612288, BD), anti-total-p38 (Cat# 9212, Cell signaling), anti-human Aβ 1-17 clone 6E10 (Cat# 9320-02, Signet), anti-synaptophysin (Cat# MAB5258; Chemicon), anti-synaptojanin 1 antibody (AC1), (Cat# MA3-936; Thermo Fisher), anti-NMDAR2B (Cat# ab81271, Abcam), anti-BACE1 antibody (Cat# ab108394, Abcam), and β-actin (Cat# A5441; Sigma-Aldrich).

Techniques: Inhibition, Injection

Journal: Nature Communications

Article Title: Overexpression of endophilin A1 exacerbates synaptic alterations in a mouse model of Alzheimer’s disease

doi: 10.1038/s41467-018-04389-0

Figure Lengend Snippet: Blocking EP-mediated oxidative stress and p38 activation rescued Aβ-induced synaptic vesicle recycling impairment. Fourteen-day in vitro cultured cortical neurons, either non-Tg or Tg Sh3gl2 , were treated with 50 nM Aβ for 24 h, with or without 500 nM EUK-134, 1 μM SB203580/SB, or 1 μM MitoTEMPO pretreatment for 1 h before the addition of Aβ. To visualize synaptic vesicle recycling, the cells were loaded with the fluorescent styryl dye FM1–43 before and after stimulation with 50 mM K + for the indicated time. a , f Kinetics of FM1–43 unloading of synaptic boutons during sustained stimulation with 50 mM KCl. b–e Fluorescence images before (I) and after (II, III) FM1–43 unloading with 50 mM KCl, and the representative immunofluorescence images of MAP2 (red, IV) to ensure the position of FM1–43 fluorescence (green, IV in d , e ). Tg Sh3gl2 neurons treated with 50 nM Aβ for 24 h alone ( e , h ) showed synaptic vesicle release impairment compared to the vehicle Tg Sh3gl2 treatment ( c , g ) and non-Tg neurons, whereas treatment with 50 nM Aβ ( d ) showed no difference compared to the vehicle non-Tg neurons ( b ). Pretreatment with 500 nM EUK-134 ( i ), 1 μM SB203580 ( j ), or 1 μM MitoTEMPO ( k ) rescued Aβ-induced synaptic vesicle recycling impairment in Tg Sh3gl2 neurons. Scale bar = 50 µm. Error bars represent s.e.m., n = 8 per group. * p < 0.01 compared to other groups in a and f (one-way ANOVA)

Article Snippet: The primary antibodies used were as follows: anti-EP (Cat# 36-3000, Invitrogen), anti-phos-p38 (Cat# 612288, BD), anti-total-p38 (Cat# 9212, Cell signaling), anti-human Aβ 1-17 clone 6E10 (Cat# 9320-02, Signet), anti-synaptophysin (Cat# MAB5258; Chemicon), anti-synaptojanin 1 antibody (AC1), (Cat# MA3-936; Thermo Fisher), anti-NMDAR2B (Cat# ab81271, Abcam), anti-BACE1 antibody (Cat# ab108394, Abcam), and β-actin (Cat# A5441; Sigma-Aldrich).

Techniques: Blocking Assay, Activation Assay, In Vitro, Cell Culture, Fluorescence, Immunofluorescence

Journal: Nature Communications

Article Title: Overexpression of endophilin A1 exacerbates synaptic alterations in a mouse model of Alzheimer’s disease

doi: 10.1038/s41467-018-04389-0

Figure Lengend Snippet: Effect of EP overexpression on cerebral Aβ accumulation. ELISA for measurement of Aβ40 ( a , c ) and Aβ42 ( b , d ) in the entorhinal cortex of Tg mAPP and Tg Sh3gl2 /mAPP mice at the age of 5–5.5 months. EUK-134 (EUK, 2 mg/kg) ( c , d ) or SB203580 (SB, 0.5 mg/kg) ( c , d ) was administered to Tg Sh3gl2 /mAPP mice once a day for 3 weeks and then cortical tissues were subjected to Aβ measurement at the age of 5–5.5 months. Date are shown as mean ± s.e.m., n = 3–6 per group (one-way ANOVA in a–d ). Quantification of immunoreactive bands for Aβ ( e ), BACE1 ( g ), or IDE ( i ) in the indicated Tg mice at the age of 5–5.5 months. Quantification of immunoreactive bands for Aβ ( f ), BACE1 ( h ), or IDE ( j ) in Tg Sh3gl2 .mAPP mice treated with EUK or P38 inhibitor (SB) relative to vehicle treatment. β-Actin was used as a protein loading control. Lower panels are representative immunoblots for the indicated proteins in the indicated Tg mice. Date are shown as mean ± s.e.m., n = 3 per group (one-way ANOVA in e–j )

Article Snippet: The primary antibodies used were as follows: anti-EP (Cat# 36-3000, Invitrogen), anti-phos-p38 (Cat# 612288, BD), anti-total-p38 (Cat# 9212, Cell signaling), anti-human Aβ 1-17 clone 6E10 (Cat# 9320-02, Signet), anti-synaptophysin (Cat# MAB5258; Chemicon), anti-synaptojanin 1 antibody (AC1), (Cat# MA3-936; Thermo Fisher), anti-NMDAR2B (Cat# ab81271, Abcam), anti-BACE1 antibody (Cat# ab108394, Abcam), and β-actin (Cat# A5441; Sigma-Aldrich).

Techniques: Over Expression, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: Distinct Mutations in IRAK-4 Confer Hyporesponsiveness to Lipopolysaccharide and Interleukin-1 in a Patient with Recurrent Bacterial Infections

doi: 10.1084/jem.20030701

Figure Lengend Snippet: Patient's PBMCs exhibit impaired p38 phosphorylation in response to LPS or IL-1. PBMCs derived from an unrelated control or the patient (A) or the patient's sister and the patient (B) were stimulated with 1 μg/ml LPS, 100 ng/ml rIL-1β, or 100 ng/ml rTNF-α for 15 min. Whole cell lysates were analyzed by Western blot analysis for phosphorylated p38 or total ERK-1,2.

Article Snippet: Abs to human IRAK-1 (Upstate Biotechnology), phosphorylated p38 (Promega), total ERK 1,2 (New England Biolabs, Inc.), and Flag (M2; Sigma-Aldrich) were used for Western blot analysis.

Techniques: Phospho-proteomics, Derivative Assay, Control, Western Blot