Journal: Molecular Vision

Article Title: Effect of curcumin on the modulation of αA- and αB-crystallin and heat shock protein 70 in selenium-induced cataractogenesis in Wistar rat pups

doi:

Figure Lengend Snippet: Effects of curcumin on immunohistochemistry of αA- and αB-crystallin in the eye lens of Wistar rat pups exposed to selenium. A : This image shows the control lens incubated in saline alone without any antibody treatment. B : This is a control lens that was incubated with saline and subjected to antibody treatment. C : Lens from rat pups administered with selenium alone. D : Lens from rat pups administered with selenium and curcumin simultaneously. E : Lens from rat pups administered with selenium first and then treated with curcumin after 24 h. F : Lens from rat pups pretreated with curcumin and then administered with selenium after 24 h. Lens sections were preincubated with αA- and αB-crystallin polyclonal antirabbit Immunoglobulin G (IgG) antibody (1:3,000 dilution) and subsequently with goat anti-rabbit IgG-horse radish peroxidase (HRP) conjugate (1:3,000 dilution). The immunoreactivity was developed with 0.01% 3,3-diaminobenzidine tetrahydrochloride (DAB) and H 2 O 2 . Note the brown color formation indicative of peroxidase reaction in the nucleus. Image G , negative control, lens treated with goat anti-rabbit IgG-HRP and developed using DAB and hydrogen peroxide (H 2 O 2 ). The figure shows the high level of αA- and αB-crystallin expression induced by selenium-mediated oxidative stress. This increased crystallin expression and aggregate formation was prevented by curcumin pretreatment.

Article Snippet: After washing with TBS containing 0.05% Tween 20, the sections were incubated with the primary antibody, αA- and αB-crystallin polyclonal anti-rabbit IgG antibody (BD Biosciences, San Jose, CA), at a dilution of 1:500 overnight at 4 °C.

Techniques: Immunohistochemistry, Incubation, Negative Control, Expressing

Journal: Molecular Vision

Article Title: Effect of curcumin on the modulation of αA- and αB-crystallin and heat shock protein 70 in selenium-induced cataractogenesis in Wistar rat pups

doi:

Figure Lengend Snippet: Immunoblot expression of αA- and αB-crystallin in control and experimental group of animals. Lane I, eye lens protein from control (physiologic saline) rat pups (group I); lane II, eye lens protein from selenium-injected rat pups (group II); lane III, eye lens protein from rat pups administered selenium and curcumin simultaneously (group III); lane IV, eye lens protein from rat pups injected with selenium 24 h before being administered with curcumin (group IV); and lane V, eye lens protein from rat pups administered with curcumin 24 h before being injected with selenium (group V). The separated lens protein was preincubated with αA- and αB-crystallin polyclonal antirabbit IgG antibody (1:3,000 dilution) and subsequently with goat antirabbit IgG-HRP (1:3,000 dilution). The immunoreactivity was developed with 0.01% DAB and H 2 O 2 . β-Actin refers to house keeping protein expression and its levels are constant across all treatment groups indicating the normal behaviour of lenses under various treatment. The figure clearly shows increased αA- and αB-crystallin protein expression under selenium-mediated oxidative stress. This increased crystallin protein expression was prevented by curcumin pretreatment.

Article Snippet: After washing with TBS containing 0.05% Tween 20, the sections were incubated with the primary antibody, αA- and αB-crystallin polyclonal anti-rabbit IgG antibody (BD Biosciences, San Jose, CA), at a dilution of 1:500 overnight at 4 °C.

Techniques: Western Blot, Expressing, Injection

Journal: bioRxiv

Article Title: Specificity and effector functions of non-neutralizing gB-specific monoclonal antibodies isolated from healthy individuals with human cytomegalovirus infection

doi: 10.1101/2020.01.14.907204

Figure Lengend Snippet: gB-specific mAbs mediate negligible NK activation ADCC responses when compared to polyclonal CMV-specific antibody preparation (Cytogam). gB-specific mAbs were screened for mediation of NK cell (A) CD107a degranulation and (B) CD16 downregulation (DRI) as measures of antibody-mediated NK cell activation. Data are presented as mAb specific response against AD169r-GFP infected ARPE19 cells minus mock infected negative control cells.

Article Snippet: Purity and identity were confirmed by Western blot using polyclonal CMV IgG (Cytogam – CSL Behring) or monoclonal antibodies SM10 (Domain I) and SM5-1 (Domain II).

Techniques: Activation Assay, Infection, Negative Control

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