Journal: bioRxiv

Article Title: Selective Immune Silencing by Targeted TGF-β Agonists

doi: 10.64898/2026.01.19.700410

Figure Lengend Snippet: Design and characterization of mouse T cell-targeted TGF- β agonists. a. Schematic of the plasmid construct encoding the MSA–TGM1-targeting-arm fusion protein. b. SDS–PAGE analysis showing the molecular weight and purity of the indicated recombinant proteins. c. SDS–PAGE analysis showing the molecular weight and purity of the indicated recombinant proteins. d. Representative flow cytometry plots showing mouse CD4 and CD8α expression in mouse CD4- or CD8α-transduced HEK293 cells. e. Dose-dependent curves showing GITR, IRF4, and T-bet expression levels in in vitro –cultured CD4 T cells treated with the indicated proteins on day 3. f. Dose-dependent curves showing GITR, IRF4, and T-bet expression levels in in vitro –cultured CD8 T cells treated with the indicated proteins on day 3. g. Dose-dependent curves showing percentages of IFN-γ– and TNF-α–producing cells among in vitro –cultured CD4 T cells treated with the indicated proteins on day 3. h. Dose-dependent curves showing percentages of IFN-γ– and TNF-α–producing cells among in vitro –cultured CD8 T cells treated with the indicated proteins on day 3. i. Dose-dependent curves showing Foxp3 cell percentages in in vitro -cultured CD4 T cells, and Granzyme B–producing cell percentages in in vitro -cultured CD8 T cells treated with the indicated proteins on day 3. Data are presented as mean ± s.e.m.

Article Snippet: The following antibodies were purchased from Miltenyi Biotec: human IgA (130-113-480) and IRF4 (130-100-909).

Techniques: Plasmid Preparation, Construct, SDS Page, Molecular Weight, Recombinant, Flow Cytometry, Expressing, In Vitro, Cell Culture

Journal: bioRxiv

Article Title: Selective Immune Silencing by Targeted TGF-β Agonists

doi: 10.64898/2026.01.19.700410

Figure Lengend Snippet: a. Frequencies of immune cell subsets. b. SCENIC prediction of IRF4 and Blimp1 transcription factor activity. c. Immunoglobulin gene expression. d. UMAP plots showing cell cycle gene signature scores. e. Quantification of cell cycle gene signature scores. f. Scaled signature scores for TGF-β–inducible genes, senescence genes, and apoptosis-related genes, aggregated by sample.

Article Snippet: The following antibodies were purchased from Miltenyi Biotec: human IgA (130-113-480) and IRF4 (130-100-909).

Techniques: Activity Assay, Gene Expression

Journal: Endocrinology

Article Title: Msx Homeobox Genes Act Downstream of BMP2 to Regulate Endometrial Decidualization in Mice and in Humans

doi: 10.1210/en.2019-00131

Figure Lengend Snippet: Msx2 is a downstream target of BMP2 signaling in the uterus during decidualization. (A) The primary cultures of mouse endometrial stromal cells (MESCs) were transduced with adenovirus expressing GFP or BMP2. The cells were lysed at different time points, as indicated. Total RNA was isolated, and real-time PCR was performed to analyze the levels of Msx2. The relative levels of gene expression were determined by setting the expression level of the GFP-treated sample at 24 h to 1.0 (n = 3). Rplp0, encoding a ribosomal protein, was used to normalize the level of RNA. *P < 0.05. (B) The nucleotide positions of the SBEs on the Msx2 promoter were analyzed by ChIP. (C) Mouse stromal cells were treated with E + P or E, P, and BMP2 (E + P + BMP2) for 90 min. ChIP, using the Smad4 antibody, was performed, as described in “Materials and Methods.” Chromatin enrichment was quantified by real-time PCR using primers flanking the potential SBE in the Msx2 promoter and also a negative control region in the ORF of Msx2. Enrichments were normalized to 1% of input DNA. The experiment was repeated twice, and representative data are shown.

Article Snippet: The next day, the cells were either treated with E + P or E + P + BMP2 (recombinant human BMP2; R&D Systems; 355-BM-010) for 90 minutes.

Techniques: Transduction, Expressing, Isolation, Real-time Polymerase Chain Reaction, Gene Expression, Negative Control

Journal: Endocrinology

Article Title: Msx Homeobox Genes Act Downstream of BMP2 to Regulate Endometrial Decidualization in Mice and in Humans

doi: 10.1210/en.2019-00131

Figure Lengend Snippet: MSX1 and MSX2 mediate BMP2-induced HESC decidualization. The primary cultures of HESCs were established as described in “Materials and Methods.” The cells were transduced with adenovirus expressing GFP or BMP2. The cells were lysed at different time points as indicated. Total RNA was isolated, and real-time PCR was performed to analyze the levels of MSX1 and MSX2. The relative levels of gene expression were determined by setting the expression level on day 0 of the GFP-treated sample at 1.0. RPLP0, encoding a ribosomal protein, was used to normalize the level of RNA. Data were collected from three independent clinical samples, which were subjected to the same experimental conditions. *P < 0.05; **P < 0.005.

Article Snippet: The next day, the cells were either treated with E + P or E + P + BMP2 (recombinant human BMP2; R&D Systems; 355-BM-010) for 90 minutes.

Techniques: Transduction, Expressing, Isolation, Real-time Polymerase Chain Reaction, Gene Expression

Journal: Bioengineering

Article Title: RNU ( Foxn1 RNU -Nude) Rats Demonstrate an Improved Ability to Regenerate Muscle in a Volumetric Muscle Injury Compared to Sprague Dawley Rats

doi: 10.3390/bioengineering8010012

Figure Lengend Snippet: CD68+ staining is increased in RNU DMM-treated sites compared to Sprague Dawley. CD68 staining was quantified and showed increased levels in RNU rats ( A ). Yet, CD163 ( B ) and FoxP3 ( C ) were unaltered. CD4 staining was increased in RNU rats ( D ) while CD8 staining was lower ( E ). In addition, CD4, CD8, and FoxP3 levels were low. DRAQ5 showed no change in DNA staining ( F ). Data shown are means ± SEM of 8 animals. Letters not shared indicated a significant difference ( p < 0.05, Tukey).

Article Snippet: Primary antibodies used in this experiment were: mouse anti-Pax7 (ab55494, Abcam, Cambridge, UK); rabbit anti-nicotinic acetylcholine receptor-epsilon (AChR-ε, ab65180, Abcam); mouse anti-nicotinic acetylcholine receptor-gamma (AChR-γ, MA3-043, Thermo Fisher Scientific, Waltham, MA, USA); mouse anti-myosin heavy chain-fetal (fMyHC, SC-53097, Santa Cruz Biotechnology); CD68 (ab125212, Abcam); CD163 (ab87099, Abcam); CD8 (MAB116, R&D Systems), CD4 (MAB554, R&D Systems), FoxP3 (MAB8214, R&D Systems) and were diluted in PBS with 1% BSA and 0.3% Tween-20.

Techniques: Staining

Journal: European Journal of Oral Sciences

Article Title: Higher expression of WNT 5A protein in oral squamous cell carcinoma compared with dysplasia and oral mucosa with a normal appearance

doi: 10.1111/eos.12352

Figure Lengend Snippet: Expression of WNT 5A in different tissue regions. (A and B) Nuclear expression of WNT5A in oral mucosa with normal appearance. (C and D) Cytoplasmic and nuclear expression of WNT5A in severe‐grade dysplasia (black arrows). (E and F) Cancer islands; black arrows indicate expression of WNT5A in the cytoplasm in the periphery of cancer islands and red arrows indicate the absence of expression of WNT5A in the central part of the cancer islands. Scale bar = 50 μ m.

Article Snippet: The cells were cultured in six‐well plates until 80% confluence was reached, rinsed with PBS, incubated with serum‐free medium overnight, rinsed again with PBS, and treated either with 0.1% BSA in PBS as the control or with 0.4 μ g/ml of recombinant WNT5A (rWNT5A; 645‐WN; R&D Systems, Minneapolis, MN, USA) in serum‐free medium.

Techniques: Expressing

Journal: European Journal of Oral Sciences

Article Title: Higher expression of WNT 5A protein in oral squamous cell carcinoma compared with dysplasia and oral mucosa with a normal appearance

doi: 10.1111/eos.12352

Figure Lengend Snippet: Effect of recombinant WNT 5A ( rWNT 5A) on expression of β ‐catenin and E‐cadherin in SCC 9 and SCC 25. (A, E) Representative western blots of expression of active β ‐catenin, total β ‐catenin, and E‐cadherin after stimulation with rWNT 5A. (B, F) Quantification of relative E‐cadherin; (C, G) quantification of active β ‐catenin; (D, H) quantification of total β ‐catenin. All quantifications were performed on four separate experiments.

Article Snippet: The cells were cultured in six‐well plates until 80% confluence was reached, rinsed with PBS, incubated with serum‐free medium overnight, rinsed again with PBS, and treated either with 0.1% BSA in PBS as the control or with 0.4 μ g/ml of recombinant WNT5A (rWNT5A; 645‐WN; R&D Systems, Minneapolis, MN, USA) in serum‐free medium.

Techniques: Recombinant, Expressing, Western Blot

Journal: European Journal of Oral Sciences

Article Title: Higher expression of WNT 5A protein in oral squamous cell carcinoma compared with dysplasia and oral mucosa with a normal appearance

doi: 10.1111/eos.12352

Figure Lengend Snippet: Expression of WNT5A, β ‐catenin, and E‐cadherin in oral mucosa with a normal appearance, dysplasia, and at the invasive front of oral squamous cell carcinoma (OSCC)

Article Snippet: The cells were cultured in six‐well plates until 80% confluence was reached, rinsed with PBS, incubated with serum‐free medium overnight, rinsed again with PBS, and treated either with 0.1% BSA in PBS as the control or with 0.4 μ g/ml of recombinant WNT5A (rWNT5A; 645‐WN; R&D Systems, Minneapolis, MN, USA) in serum‐free medium.

Techniques: Expressing, Staining

Journal: Development (Cambridge, England)

Article Title: Evolutionarily conserved requirement of Cdx for post-occipital tissue emergence.

doi: 10.1242/dev.079848

Figure Lengend Snippet: Fig. 5. Addition of Fgf8 to the whole embryo culture medium of Cdx2 null embryos rescues their axial elongation. (A,B)E8.0 (early somite) embryos at the start of the culture (A, wild type; B, Cdx2 null). (C-F)Embryos after their culture for the same period without (C,D) or with (E,F) Fgf8 added to the culture medium. C and E are controls; D and F are Cdx2 null mutants. (G-J)Mox1 expression in another set of Cdx2 null mutants (H,J) and controls (G,I) that have been cultured for the same period with (I,J) and without (G,H) Fgf8. (K)Comparison of posterior elongation of Cdx2 null and control embryos cultured without and with Fgf8. y axis, total number of somites generated in culture; bars on the graph represent median values; n, number of embryos; several experimental data are superimposed as one symbol in the graph as they had the same value. (L)Statistical analysis of the axial growth rescue of Cdx mutants by Fgf8 using the Mann-Whitney U test. al, allantois; flb, forelimb bud. Anterior is to the right in A,B and up in C-J. Scale bars: 0.5 mm. See also supplementary material Fig. S3.

Article Snippet: Recombinant Fgf8 (isoform b) was purchased from R&D Systems (423- F8).

Techniques: Embryo Culture, Expressing, Cell Culture, Comparison, Control, Generated, MANN-WHITNEY

Journal: American journal of physiology. Endocrinology and metabolism

Article Title: Myostatin propeptide mutation of the hypermuscular Compact mice decreases the formation of myostatin and improves insulin sensitivity.

doi: 10.1152/ajpendo.00216.2016

Figure Lengend Snippet: Fig. 3. Myostatin level in skeletal muscle of Compact, congenic wild-type, and BALB/c mice. M. gastrocnemius protein extracts were subjected to SDS-PAGE and blotted with anti-myostatin or anti-propeptide antibody. Represen- tative images are shown. Note the presence of mature myostatin dimer and myostatin propeptide in Compact sam- ples. Mouse recombinant myostatin was used as a positive control, and muscle homogenates of myostatin knockout (KO) mice served as a negative control. Differences in glycosylation may cause altered electrophoretic mobility. Bar diagrams show the quantification of the results. Data are reported as means SE; n 5 Compact, 5 congenic wild-type, and 6 BALB/c mice. *P 0.05; **P 0.01; ***P 0.001.

Article Snippet: Mouse recombinant myostatin (788-G8; R&D Systems) was used as a positive control.

Techniques: SDS Page, Recombinant, Positive Control, Knock-Out, Negative Control, Glycoproteomics

Journal: American journal of physiology. Endocrinology and metabolism

Article Title: Myostatin propeptide mutation of the hypermuscular Compact mice decreases the formation of myostatin and improves insulin sensitivity.

doi: 10.1152/ajpendo.00216.2016

Figure Lengend Snippet: Fig. 5. Glucose tolerance and insulin sensitivity are improved by Compact myostatin mutation and reduced in congenic wild-type mice. Intraperitoneal (ip) glucose tolerance (A and B) and insulin sensitivity tests (C and D) of 3- to 4-mo-old (A and C) and 10-mo-old animals (B and D). Area under the curve (AUC) values are presented in bar diagrams. Data are reported as means SE. *P 0.05 and **P 0.01; n 3 Compact, 7 congenic wild-type, and 3 BALB/c mice (A), n 7 Compact, 3 congenic wild-type, and 6 BALB/c mice (B), n 3 Compact, 3 congenic wild-type, and 4 BALB/c mice (C), and n 6 Compact, 4 congenic wild-type, and 4 BALB/c mice (D).

Article Snippet: Mouse recombinant myostatin (788-G8; R&D Systems) was used as a positive control.

Techniques: Mutagenesis

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