Journal: BMC Neuroscience

Article Title: Chronic exposure to high fat diet triggers myelin disruption and interleukin-33 upregulation in hypothalamus

doi: 10.1186/s12868-019-0516-6

Figure Lengend Snippet: List of antibodies in the study for immunofluorescence or western blot analysis

Article Snippet: Polyclonal goat anti-IL-33 , R and D Systems Cat#AF3626, RRID:AB_884269 , Escherichia coli -derived recombinant mouse IL-33 , 1:200 (IF).

Techniques: Immunofluorescence, Western Blot, Purification, Affinity Chromatography, Recombinant, Derivative Assay

Journal: Journal of Cellular Physiology

Article Title: Statin regulated ERK5 stimulates tight junction formation and reduces permeability in human cardiac endothelial cells

doi: 10.1002/jcp.26064

Figure Lengend Snippet: siRNA mediated gene silencing of ERK5 induced HCMEC barrier perturbment. HCMEC were transfected with non‐silencing or ERK5 siRNA for 6 hr and allowed to reach confluence over 5 subsequent days. (a) Immunofluorescence imaging of tight junctions (ZO‐1, green), actin stress fibers (phalloidin, red), and nuclei (Hoechst, blue). Arrows indicate barrier perturbment. Scale bars: 10 μm. Results are from one experiment representative of three. (b) Western blot of ZO‐1, ERK5, ERK1/2, and GAPDH levels in HCMECs following siRNA induced ERK5 gene silencing. (c) HCMECs were plated on Thincerts™ containing 0.4 μm pores and permeability of 4 kDa FITC‐dextran across the HCMEC monolayer was assessed and compared to untransfected cells ( n = 4), mean ± s.d. ** p ≤ 0.01 compared to untransfected. (d) Assessment of TEER was conducted on HCMECs plated on Thincerts™ containing 0.4 μm pores ( n = 4), mean ± s.d. ** p ≤ 0.01 compared to untransfected

Article Snippet: For immunoprecipitation, lysates were incubated with ERK5 (2 μg/ml, #AF2848, R&D systems) or control IgG (2 μg/ml, goat) and protein G agarose.

Techniques: Transfection, Immunofluorescence, Imaging, Western Blot, Permeability

Journal: Journal of Cellular Physiology

Article Title: Statin regulated ERK5 stimulates tight junction formation and reduces permeability in human cardiac endothelial cells

doi: 10.1002/jcp.26064

Figure Lengend Snippet: Adenoviral mediated ERK5 expression stimulates HCMEC tight junction formation. HCMEC were transfected with Ad‐Control or Ad‐CA‐MEK5 and Ad‐ERK5 for 24 hr before treatment with doxorubicin 0.1 μM for 6 hr. (a) Immunofluorescence imaging of HCMEC tight junctions (ZO‐1, green), actin stress fibers (phalloidin, red), and nuclei (Hoechst, blue). Arrows indicate barrier perturbment. Scale bars: 10 μm. Results are from one experiment representative of three. (b) Western blot of ERK5, ERK1/2, phospho ERK1/2, and GAPDH levels in HCMECs. FLAG‐Tag was used to confirm transfection of Ad‐ERK5 and HA‐Tag was used to confirm transfection of Ad‐CA‐MEK5. (c) HCMECs were plated on Thincerts™ with 0.4 μm pores and permeability of 4 kDa FITC‐dextran across the HCMEC monolayer assessed and compared to Ad‐Control ( n = 4), mean ± s.d. * p ≤ 0.05 compared to Ad‐Control

Article Snippet: For immunoprecipitation, lysates were incubated with ERK5 (2 μg/ml, #AF2848, R&D systems) or control IgG (2 μg/ml, goat) and protein G agarose.

Techniques: Expressing, Transfection, Control, Immunofluorescence, Imaging, Western Blot, FLAG-tag, Permeability

Journal: Journal of Cellular Physiology

Article Title: Statin regulated ERK5 stimulates tight junction formation and reduces permeability in human cardiac endothelial cells

doi: 10.1002/jcp.26064

Figure Lengend Snippet: Simvastatin stimulates ERK5 phosphorylation in HCMECs. (a) HCMECs were incubated with simvastatin at a range of concentrations for 6 hr. Intracellular signaling responses were assessed by Western blotting for phosphorylation of ERK5 and ERK1/2 using phospho‐specific antibodies. Unprenylation of Rap1A was measured as a control for simvastatin activity. Total protein was measured for ERK5, ERK1/2, and Rap1. (b) HCMECs were incubated with 0.3 μM simvastatin at a range of time points. Intracellular signaling responses were assessed by Western blotting for phosphorylation of ERK5 and ERK1/2 using phospho‐specific antibodies. Unprenylation of Rap1 was measured as a control for simvastatin activity. Total protein was measured for ERK5, ERK1/2, and Rap1. (c) The level of ERK5 band shift following treatment with simvastatin, rosuvastatin, and pitavastatin was quantified relative to vehicle control. (d) Activation of ERK5 by band shift was determined (EC 50 concentration), mean ± s.d. ( n = 3). (e) HCMEC were transfected with siRNA to MEKK2, MEKK3, MEK5, and ERK5 for 6 hr and allowed to reach confluence over 5 subsequent days before treatment with 0.1% DMSO or 0.3 μM simvastatin for 6 hr. Western blot of ERK5, phosphorylated‐ERK5, MEKK2, MEKK3, MEK5, and GAPDH levels in HCMEC. (f) Quantification of ERK5 band shift ( n = 3) mean ± s.d. * p ≤ 0.05 compared to untransfected basal

Article Snippet: For immunoprecipitation, lysates were incubated with ERK5 (2 μg/ml, #AF2848, R&D systems) or control IgG (2 μg/ml, goat) and protein G agarose.

Techniques: Phospho-proteomics, Incubation, Western Blot, Control, Activity Assay, Electrophoretic Mobility Shift Assay, Activation Assay, Concentration Assay, Transfection

Journal: Journal of Cellular Physiology

Article Title: Statin regulated ERK5 stimulates tight junction formation and reduces permeability in human cardiac endothelial cells

doi: 10.1002/jcp.26064

Figure Lengend Snippet: Statin induced ERK5 phosphorylation occurs via inhibition of protein geranylgeranylation. (a) Summary of the key substrates in the cholesterol biosynthesis pathway. HCMECs were treated with (b) mevalonolactone 50 μM, GGPP 10 μM, FPP 10 μM, squalene 10 μM, or cholesterol 10 μM for 24 hr in the presence and absence of simvastatin (0.3 μM for 6 hr). ERK5 expression was assessed by Western blotting. Unprenylation of Rap1A was measured as a control for simvastatin activity. The level of ERK5 activation assessed by mobility band shift was quantified and expressed as fold change relative to vehicle control ( n = 4) mean ± s.d. * p ≤ 0.05, ** p ≤ 0.01 compared to vehicle control basal. (c) HCMECs were treated with GGTI‐298 (10 μM) for a range of time points. Intracellular signaling responses were assessed by Western blotting for ERK5, unprenylated Rap1A and Rap1. (d) Immunofluorescence imaging of HCMEC tight junctions (ZO‐1, green), actin stress fibers (phalloidin, red), and nuclei (Hoechst, Blue) following treatment with GGTI‐298 (10 μM for 6 hr). Scale bars: 10 μm. (e) HCMEC were plated on Thincerts™ with 0.4 μm pores and permeability of 4 kDa FITC‐dextran across the HCMEC monolayer was assessed ( n = 4), mean ± s.d. * p ≤ 0.05 compared to vehicle control

Article Snippet: For immunoprecipitation, lysates were incubated with ERK5 (2 μg/ml, #AF2848, R&D systems) or control IgG (2 μg/ml, goat) and protein G agarose.

Techniques: Phospho-proteomics, Inhibition, Expressing, Western Blot, Control, Activity Assay, Activation Assay, Electrophoretic Mobility Shift Assay, Immunofluorescence, Imaging, Permeability

Journal: Journal of Cellular Physiology

Article Title: Statin regulated ERK5 stimulates tight junction formation and reduces permeability in human cardiac endothelial cells

doi: 10.1002/jcp.26064

Figure Lengend Snippet: MEK5 inhibition prevents simvastatin induced tight junction formation and ERK5 phosphorylation. HCMECs were pre‐incubated with BIX02189 1 μM for 30 min priopr to addition of simvastatin 0.3 μM for 6 hr. (a) Immunofluorescence imaging of HCMEC tight junctions (ZO‐1, green), actin stress fibers (phalloidin, red), and nuclei (Hoechst, blue). Scale bars: 10 μm. Results are from one experiment representative of three. (b) Western blot of ZO‐1, ERK5, phospho ERK5, unprenylated Rap1A, and GAPDH levels in HCMECs. Level of ERK5 phosphorylation is quantified relative to vehicle control. Mean ± s.d. ( n = 3) * p ≤ 0.05 compared to vehicle control basal. (c) Permeability of 4 kDa FITC‐dextran through an endothelial monolayer on ThinCerts™, ( n = 4), mean ± s.d. * p ≤ 0.05, ** p ≤ 0.01 compared to vehicle control. (d) Assessment of TEER was conducted on HCMECs plated on Thincerts™ containing 0.4 μm pores ( n = 4), mean ± s.d. ** p ≤ 0.01 compared to vehicle control. (e) HCMECs were treated with simvastatin 0.3 μM for 6 hr before immunoprecipitation with IgG or ERK5 antibodies. Western blot of ZO‐1 following immunoprecipitation with ERK5 in HCMECs. (f) Level of ZO‐1 and ERK5 is quantified relative to vehicle control in IP lysates. Mean ± s.d. ( n = 3). ** p ≤ 0.01, compared to vehicle control

Article Snippet: For immunoprecipitation, lysates were incubated with ERK5 (2 μg/ml, #AF2848, R&D systems) or control IgG (2 μg/ml, goat) and protein G agarose.

Techniques: Inhibition, Phospho-proteomics, Incubation, Immunofluorescence, Imaging, Western Blot, Control, Permeability, Immunoprecipitation

Journal: Journal of Cellular Physiology

Article Title: Statin regulated ERK5 stimulates tight junction formation and reduces permeability in human cardiac endothelial cells

doi: 10.1002/jcp.26064

Figure Lengend Snippet: Simvastatin prevents doxorubicin induced barrier perturbment. HCMEC were pre‐incubated with simvastatin 0.3 μM for 6 hr before treatment with doxorubicin 0.1 μM for a further 6 hr. (a) Immunofluorescence imaging of HCMEC tight junctions (ZO‐1, green), actin stress fibers (phalloidin, red), and nuclei (Hoechst, blue). Scale bars: 10 μm. Results are from one experiment representative of three. (b) Western blot of ZO‐1, ERK5, ERK1/2, unprenylated Rap1A and Rap1 levels in HCMECs. Level of ERK5 activation is quantified relative to vehicle control. Mean ± s.d. ( n = 3) ** p ≤ 0.01 compared to vehicle control. (c) HCMEC were plated on Thincerts™ with 0.4 μm pores and permeability of 4 kDa FITC‐dextran across the HCMEC monolayer was assessed and compared to vehicle control ( n = 4), mean ± s.d. * p ≤ 0.05, ** p ≤ 0.01 compared to vehicle control. (d) Assessment of TEER was conducted on HCMECs plated on Thincerts™ with 0.4 μm pores ( n = 4), mean ± s.d. * p ≤ 0.05, ** p ≤ 0.01 compared to vehicle control

Article Snippet: For immunoprecipitation, lysates were incubated with ERK5 (2 μg/ml, #AF2848, R&D systems) or control IgG (2 μg/ml, goat) and protein G agarose.

Techniques: Incubation, Immunofluorescence, Imaging, Western Blot, Activation Assay, Control, Permeability

Journal: Journal of Cellular Physiology

Article Title: Statin regulated ERK5 stimulates tight junction formation and reduces permeability in human cardiac endothelial cells

doi: 10.1002/jcp.26064

Figure Lengend Snippet: ERK5 and ZO‐1 colocalize following simvastatin treatment in HCMECs. Immunofluorescence imaging of HCMEC tight junctions (ZO‐1, green), ERK5 (red), and nuclei (Hoechst, blue) following treatment with simvastatin 0.3 μM and/or doxorubicin 0.1 μM or BIX02189 1 μM for 6 hr. Scale bars: 10 μm. Arrows indicate co‐localization. Results are from one experiment representative of three

Article Snippet: For immunoprecipitation, lysates were incubated with ERK5 (2 μg/ml, #AF2848, R&D systems) or control IgG (2 μg/ml, goat) and protein G agarose.

Techniques: Immunofluorescence, Imaging

Journal: Journal of Cellular Physiology

Article Title: Statin regulated ERK5 stimulates tight junction formation and reduces permeability in human cardiac endothelial cells

doi: 10.1002/jcp.26064

Figure Lengend Snippet: ERK5 cellular translocation following simvastatin treatment in HCMECs. HCMECs were treated with simvastatin 0.3 μM for 6 hr before subcellular fractionation performed. (a) Western blot of ZO‐1 and ERK5. ATP1A1, actin, and lamin B were used as positive controls to show cellular fraction specificity in membrane, cytoplasm, and nuclear fractions respectively. Whole cell lysate (WCL). (b) Quantification of the total ERK5 level in each compartment ( n = 3), mean ± s.d. * p ≤ 0.05 compared to vehicle control for each fraction. (c) Quantification of ERK5 band shift relative to each fraction's vehicle control ( n = 3), mean ± s.d. * p ≤ 0.05 compared to vehicle control for each fraction

Article Snippet: For immunoprecipitation, lysates were incubated with ERK5 (2 μg/ml, #AF2848, R&D systems) or control IgG (2 μg/ml, goat) and protein G agarose.

Techniques: Translocation Assay, Fractionation, Western Blot, Membrane, Control, Electrophoretic Mobility Shift Assay