Journal:

Article Title: Compartmentalized signaling by GPI-anchored ephrin-A5 requires the Fyn tyrosine kinase to regulate cellular adhesion

doi:

Figure Lengend Snippet: Ephrin-A5 is compartmentalized within discrete plasma membrane microdomains. (A) NIH-3T3 cells stably expressing human ephrin-A5 were subjected to immunofluorescence using the Fc fragment of human IgG as negative control (a), the EphA5–Fc fusion protein (8 μg/ml) to detect the ephrin-A5 protein (b), or caveolin-1-specific monoclonal antibody (c). (B) NIH-3T3 cells ectopically expressing ephrin-A5 (A5-1) or wild type (WT) were lysed in 1% Triton X-100, and the caveolar (C) and Triton X-100 soluble (S) fractions were collected as described previously (Robbins et al. 1995) (left). The isolated caveolae-like fraction from ephrin-A5-expressing cells was incubated with 5 μg of either EphA5–Fc or Fc fusion proteins, pelleted by the addition of protein A–Sepharose and analyzed by immunoblotting (right). Both blots were probed with a monoclonal antibody to ephrin-A5 (clone 5G2). (C) The ephrin-A5-expressing fibroblasts were lysed in 1% Triton X-100 and fractionated by linear sucrose gradients (5%–40%) as above, except individual 1-ml aliquots were collected and subjected to a Western blot analysis using caveolin-1, caveolin-2, and ephrin-A5-specific antibodies as indicated. Molecular mass standards are labeled (kD).

Article Snippet: NIH-3T3 cells were transfected with 10 μg of ephrin-A5 expression vector and either 1 μg of LNCX vector or 1 μg of pBabepuro using calcium phosphate precipitation (Canadian Life Technologies, GIBCO); individual clones were selected using 600 μg/ml G418 or 2 μg/ml puromycin, respectively.

Techniques: Stable Transfection, Expressing, Immunofluorescence, Negative Control, Isolation, Incubation, Western Blot, Labeling

Journal:

Article Title: Compartmentalized signaling by GPI-anchored ephrin-A5 requires the Fyn tyrosine kinase to regulate cellular adhesion

doi:

Figure Lengend Snippet: Ephrin-A5 is competent to transmit a compartmentalized signal event on binding to its cognate receptor. (A) NIH-3T3 cells expressing ephrin-A5 were incubated for the times indicated with either Fc or EphA5–Fc. Cells were lysed and fractionated on a sucrose gradient. Both caveolae-like fractions (C) and soluble fractions (S) were analyzed by Western blot with the 4G10 antibody. The blot was reprobed with an anti-caveolin-2 antibody to control for the amount of TX-100 insoluble material loaded. (B) Caveolae-like fractions purified from NIH-3T3 cells expressing ephrin-A5 that were stimulated with EphA5–Fc for the times indicated (minutes) were analyzed by Western blotting with the 4G10 antibody. The blot was reprobed with anti-caveolin-2 antibody to control for the amount of material loaded. Molecular mass markers are indicated in kD.

Article Snippet: NIH-3T3 cells were transfected with 10 μg of ephrin-A5 expression vector and either 1 μg of LNCX vector or 1 μg of pBabepuro using calcium phosphate precipitation (Canadian Life Technologies, GIBCO); individual clones were selected using 600 μg/ml G418 or 2 μg/ml puromycin, respectively.

Techniques: Binding Assay, Expressing, Incubation, Western Blot, Purification

Journal:

Article Title: Compartmentalized signaling by GPI-anchored ephrin-A5 requires the Fyn tyrosine kinase to regulate cellular adhesion

doi:

Figure Lengend Snippet: Fyn tyrosine kinase is recruited to the caveolae-like domains upon ephrin-A5 engagement. (A) NIH-3T3 cells expressing ephrin-A5 were incubated for different times with EphA5–Fc, in the absence or presence of PP1 as indicated. Caveolae-like fractions (cav) were isolated and analyzed by Western blot using in parallel 4G10 and an antibody specific for Fyn. (B) NIH-3T3 cells expressing ephrin-A5 were fractionated on a sucrose gradient. Caveolae-like fractions (C) and soluble fractions (S) were tested by Western blot for the presence of Fyn and Src proteins. Molecular weight mass are indicated in kD.

Article Snippet: NIH-3T3 cells were transfected with 10 μg of ephrin-A5 expression vector and either 1 μg of LNCX vector or 1 μg of pBabepuro using calcium phosphate precipitation (Canadian Life Technologies, GIBCO); individual clones were selected using 600 μg/ml G418 or 2 μg/ml puromycin, respectively.

Techniques: Expressing, Incubation, Isolation, Western Blot, Molecular Weight

Journal:

Article Title: Compartmentalized signaling by GPI-anchored ephrin-A5 requires the Fyn tyrosine kinase to regulate cellular adhesion

doi:

Figure Lengend Snippet: p80 is a putative substrate for Src-family kinases. (A) Caveolae-like fractions isolated from EphA5–Fc-stimulated fibroblasts were solubilized in TX-100 at 37°C for 30 min and incubated with 50 μg of either purified GST or GST–Src–SH2 fusion proteins. Protein complexes were pelleted by low-speed centrifugation and analyzed by Western blotting using the phosphotyrosine-specific antibody 4G10. (B) Caveolar fractions were isolated from ephrin-A5-expressing cells that had been treated with either Fc or EphA5–Fc in the presence or absence of PP1 (as indicated). Samples were analyzed as above with GST–Src–SH2 fusion protein and Western blotting with 4G10. Molecular mass markers are indicated (kD).

Article Snippet: NIH-3T3 cells were transfected with 10 μg of ephrin-A5 expression vector and either 1 μg of LNCX vector or 1 μg of pBabepuro using calcium phosphate precipitation (Canadian Life Technologies, GIBCO); individual clones were selected using 600 μg/ml G418 or 2 μg/ml puromycin, respectively.

Techniques: Isolation, Incubation, Purification, Centrifugation, Western Blot, Expressing

Journal:

Article Title: Compartmentalized signaling by GPI-anchored ephrin-A5 requires the Fyn tyrosine kinase to regulate cellular adhesion

doi:

Figure Lengend Snippet: Binding of EphA5–Fc to ephrin-A5-expressing cells induces changes in cell architecture, requiring the Src-family kinases. (A) NIH-3T3 cells expressing ephrin-A5 (A5-2) (a,b) and primary murine astrocytes (c,d) were incubated with Fc (a,c) or EphA5–Fc (b,d) proteins for 30 min at 37°C, fixed and subjected to indirect immunofluorescence using anti-vinculin monoclonal antibody (clone VIN-11-5, Sigma) and a Cy3-conjugated anti-mouse secondary antibody. (B) NIH-3T3 cells expressing ephrin-A5 (A5-2) (a–f) were incubated with Fc (a,c,e) or EphA5–Fc (b,d,f) proteins for 30 min at 37°C, in the presence of either vehicle (DMSO) (a,b), 1 μm Gö 6983 (c,d), or 10 μm PP1 (e,f). The cells were then fixed with methanol and subjected to indirect immunofluorescence using anti-vinculin monoclonal antibody.

Article Snippet: NIH-3T3 cells were transfected with 10 μg of ephrin-A5 expression vector and either 1 μg of LNCX vector or 1 μg of pBabepuro using calcium phosphate precipitation (Canadian Life Technologies, GIBCO); individual clones were selected using 600 μg/ml G418 or 2 μg/ml puromycin, respectively.

Techniques: Binding Assay, Expressing, Incubation, Immunofluorescence

Journal:

Article Title: Compartmentalized signaling by GPI-anchored ephrin-A5 requires the Fyn tyrosine kinase to regulate cellular adhesion

doi:

Figure Lengend Snippet: Activation of ephrin-A5 results in an increase in cellular adhesion. Cells expressing ephrin-A5 (A5–2 and NG108–15) or cells transfected with the vector control (WT) were detached from their substratum by nonenzymatic removal, resuspended in low-serum containing media and spun down on plates coated with fibronectin. Cells were immediately incubated with either Fc (shaded bars) or EphA–Fc (solid bars) as indicated. Nonadhering cells were removed and the remaining adhering cells were trypsinized and counted. Values are expressed as the number of adhering cells from three different experiments (in triplicate).

Article Snippet: NIH-3T3 cells were transfected with 10 μg of ephrin-A5 expression vector and either 1 μg of LNCX vector or 1 μg of pBabepuro using calcium phosphate precipitation (Canadian Life Technologies, GIBCO); individual clones were selected using 600 μg/ml G418 or 2 μg/ml puromycin, respectively.

Techniques: Activation Assay, Expressing, Transfection, Plasmid Preparation, Incubation

Journal:

Article Title: Compartmentalized signaling by GPI-anchored ephrin-A5 requires the Fyn tyrosine kinase to regulate cellular adhesion

doi:

Figure Lengend Snippet: Model for the activation of ephrin-A ligands regulating cellular architecture. Ephrin-A5 is localized to discrete membrane microdomains. Upon binding to its cognate Eph receptor expressed on a neighboring cell, a signal is initiated within the ephrin-A5-expressing cell. This signal, presumably requiring the Rho family of small molecular weight GTPases, results in a change in cellular adhesion that may affect neuronal migration and guidance. The unknown p80 phosphoprotein, the Src-family kinase Fyn, and a putative transmembrane adapter protein (X) are indicated and presumably perform some essential functions during intracellular signaling.

Article Snippet: NIH-3T3 cells were transfected with 10 μg of ephrin-A5 expression vector and either 1 μg of LNCX vector or 1 μg of pBabepuro using calcium phosphate precipitation (Canadian Life Technologies, GIBCO); individual clones were selected using 600 μg/ml G418 or 2 μg/ml puromycin, respectively.

Techniques: Activation Assay, Binding Assay, Expressing, Molecular Weight, Migration

Journal: The Journal of Experimental Medicine

Article Title: RAGE limits regeneration after massive liver injury by coordinated suppression of TNF-α and NF-κB

doi: 10.1084/jem.20040934

Figure Lengend Snippet: Up-regulation of RAGE in hepatic remnants after massive (85%) hepatectomy: cellular localization and impact of ligand/RAGE blockade. Male C57BL/6 mice were subjected to partial (70%) or massive (85%) hepatectomy or sham surgery. Mice subjected to 85% resection were treated with murine sRAGE, 100 μg per day, or vehicle, PBS daily until death. (A) Kaplan-Meier Product Limit Estimate. The times of death were recorded for mice undergoing hepatectomy, and survival was plotted. (B) Quantitative PCR. Quantitative PCR was performed on liver remnants retrieved from mice undergoing sham, 70% or 85% liver resection, or 85% resection in the presence of sRAGE. After normalization to internal controls, levels of murine RAGE transcripts in sham-treated mice livers were arbitrarily defined as “1.” In this experiment, n = 5 mice per group. *, P < 0.05 versus 70% hepatectomy. **, P < 0.05 versus PBS treatment/85% hepatectomy. (C–F) Immunofluorescence microscopy. Immunofluorescence for detection of RAGE antigen was performed in sections prepared 8 h after sham surgery (C) or massive resection (D, 85%). In tissues prepared from remnants after massive resection, double immunofluorescence staining was performed with anti-RAGE IgG (green) and an anti-CD68 IgG (E, red), or with anti-RAGE IgG (green) and anti-CD11c IgG (F, red) as described before. Single and merged images are shown. (C, E, and F) Bars, 50 μm. (G) Kaplan-Meier Product Limit Estimate. Male C57BL/6 mice were subjected to massive hepatic resection and treated with the indicated F(ab′)2 fragments of rabbit anti-RAGE, anti-S100/calgranulin, antiamphoterin, or nonimmune IgG. The times of death were recorded for mice undergoing hepatectomy, and survival was plotted.

Article Snippet: In brief, after blocking with normal serum, sections were incubated with rabbit anti-RAGE IgG (1:50 dilution; reference 40) and incubated with a biotinylated goat anti–rabbit IgG (1:100; Vector Laboratories) followed by incubation with streptavidin-conjugated to Alexa Fluor 555 green (1 μg/ml; Molecular Probes, Inc.).

Techniques: Real-time Polymerase Chain Reaction, Immunofluorescence, Microscopy, Double Immunofluorescence Staining

Journal: The Journal of Experimental Medicine

Article Title: RAGE limits regeneration after massive liver injury by coordinated suppression of TNF-α and NF-κB

doi: 10.1084/jem.20040934

Figure Lengend Snippet: Administration of sRAGE improves survival and enhances regeneration after massive hepatectomy. Male C57BL/6 mice were subjected to 85% hepatectomy in the presence of the indicated once daily dose of sRAGE or vehicle, PBS. (A) Kaplan-Meier Product Limit Estimate. The times of death were recorded for mice undergoing hepatectomy in the presence of sRAGE versus vehicle, and survival was plotted. Survival curves for sRAGE, 100 μg/d- and PBS-treated mice, reflect the same animals as in A. (B–D) Indices of injury and regeneration. At the indicated times, plasma or hepatic remnant was retrieved and analyzed for ALT (B) and Prothrombin Time (C). (D) Liver weight/body weight ratios are shown. (B and C) n = 3–8 mice per condition. (D) n = 10 (PBS) or n = 27 (sRAGE) mice per condition. (B–D) *, P < 0.05. (E and F) Histology. Hepatic remnants were retrieved and grading was performed based on the percentage of necrosis as described before; mean ± standard error is shown. (E) n = 5 mice per condition. *, P < 0.05. (F) Representative sections from PBS- and sRAGE-treated mice at 24 h are illustrated. Bar, 80 μm (inset) 160 μm. (G) Immunoprecipitation. 16 h after 85% hepatectomy, plasma from sRAGE- or PBS-treated mice was retrieved and subjected to immunoprecipitation using rabbit anti-RAGE IgG. Immunoprecipitated material was subjected to blotting using anti-S100/calgranulin IgG. Plasma was pooled from n = 3 sRAGE- or n = 3 PBS-treated plasma for immunoprecipitation studies.

Article Snippet: In brief, after blocking with normal serum, sections were incubated with rabbit anti-RAGE IgG (1:50 dilution; reference 40) and incubated with a biotinylated goat anti–rabbit IgG (1:100; Vector Laboratories) followed by incubation with streptavidin-conjugated to Alexa Fluor 555 green (1 μg/ml; Molecular Probes, Inc.).

Techniques: Immunoprecipitation

Journal: The Journal of Experimental Medicine

Article Title: RAGE limits regeneration after massive liver injury by coordinated suppression of TNF-α and NF-κB

doi: 10.1084/jem.20040934

Figure Lengend Snippet: Blockade of RAGE modulates activation of NF-κB after massive hepatectomy. (A) EMSA. Nuclear extracts were prepared from the remnants of the indicated mice and the EMSA was performed. The illustrated bands are representative of n = 4–6 mice per condition. **, P < 0.05 versus PBS treatment/85% resection. (B) Supershift assay. Remnants retrieved from mice undergoing 85% hepatectomy in the presence of sRAGE at 2 h were retrieved and subjected to incubation with anti-p50, anti-p65, or both anti-p50 and anti-p65 IgG before EMSA. (C) Immunohistochemistry. Hepatic remnants at 2 and 8 h were retrieved and subjected to immunohistochemistry with anti-p65 NF-κB subunit antibodies. Bar, 50 μm. (D) Proliferation. Hepatic remnants were retrieved and subjected to immunohistochemistry with anti-PCNA IgG and mean numbers of PCNA + cells were determined from n = 10 fields per section/mouse. The indicated results are representative of n = 5 mice per condition. Bars, 80 μm. (inset) 160 μm. (E) Western blotting of hepatic remnant lysates was performed at the indicated times using anti-cyclin D1 IgG. The illustrated bands are representative of n = 3–4 mice per condition. (F) Apoptosis. Hepatic remnants were retrieved and subjected to TUNEL assay and mean numbers of TUNEL + cells determined from n = 10 fields per section/mouse. The indicated results are representative of n = 5 mice per condition. Bar, 40 μm. (G) Western blotting on remnants was performed using anti-activated caspase-3 IgG. The illustrated bands are representative of n = 5 mice per condition. In all cases, bands were scanned into a densitometer and relative pixel units of band density reported. *, P < 0.05 versus PBS/85% resection.

Article Snippet: In brief, after blocking with normal serum, sections were incubated with rabbit anti-RAGE IgG (1:50 dilution; reference 40) and incubated with a biotinylated goat anti–rabbit IgG (1:100; Vector Laboratories) followed by incubation with streptavidin-conjugated to Alexa Fluor 555 green (1 μg/ml; Molecular Probes, Inc.).

Techniques: Activation Assay, Incubation, Immunohistochemistry, Western Blot, TUNEL Assay

Journal: The Journal of Experimental Medicine

Article Title: RAGE limits regeneration after massive liver injury by coordinated suppression of TNF-α and NF-κB

doi: 10.1084/jem.20040934

Figure Lengend Snippet: Transgenic mice expressing DN RAGE in cells of MP lineage display enhanced survival and liver regeneration after massive hepatectomy. Transgenic mice expressing human DN RAGE selectively in cells of MP lineage using the scavenger receptor type A promoter were used for these studies. (A–D) Immunofluorescence microscopy. Immunofluorescence for detection of RAGE antigen was performed in sections prepared from the remnants of transgenic mice 8 h after sham surgery (A) or massive resection (B, 85%). Double immunofluorescence staining was performed on sections prepared from transgenic mice 8 h after massive resection using the following antibodies: anti-RAGE IgG (C and D, green) and anti-CD68 IgG (C, red) or anti-CD11c IgG (D, red). Single and merged images are shown. (A and B) Bars, 100 μm; (C and D) Bars, 50 μm. (E) Kaplan-Meier Product Limit Estimate. The times of death were recorded for transgenic versus littermate control mice undergoing massive hepatectomy and survival was plotted. (F and G) Quantitative PCR. Massive hepatectomy was performed in transgenic (DN) and wild-type littermate mice (WT). Hepatic remnants were retrieved at 8 h after resection and subjected to quantitative PCR for detection of transcripts for TNF-α (F) and IL-6 (G). After normalization to internal controls, fold changes relative to WT mice remnant transcript levels (“1.0”) are reported. n = 5 mice per condition. *, P < 0.05 versus WT mice. (H) EMSA. Massive hepatectomy was performed in transgenic (DN) and wild-type littermate mice (WT). Remnants were retrieved at 2 and 8 h, and nuclear extracts were prepared for EMSA. Bands were scanned into a densitometer and relative pixel units of band density reported. *, P < 0.05 versus WT mice/85% resection. (I) Western blotting. At the indicated times after massive resection, hepatic remnants were retrieved and subjected to Western blotting using anti-cyclin D1 IgG. (H and I) The illustrated bands are representative of n = 3–4 mice per condition.

Article Snippet: In brief, after blocking with normal serum, sections were incubated with rabbit anti-RAGE IgG (1:50 dilution; reference 40) and incubated with a biotinylated goat anti–rabbit IgG (1:100; Vector Laboratories) followed by incubation with streptavidin-conjugated to Alexa Fluor 555 green (1 μg/ml; Molecular Probes, Inc.).

Techniques: Transgenic Assay, Expressing, Immunofluorescence, Microscopy, Double Immunofluorescence Staining, Real-time Polymerase Chain Reaction, Western Blot

Journal: Investigative Ophthalmology & Visual Science

Article Title: Advanced Glycation End Products and Receptor (RAGE) Promote Wound Healing of Human Corneal Epithelial Cells

doi: 10.1167/iovs.61.3.14

Figure Lengend Snippet: Involvement of the coupled RAGE ligand/receptor in human corneal epithelial (HCE) cell wound healing. Representative images of scratch assays performed on HCE cells (left panel) treated with HMGB1 (high mobility group box 1, 100 ng/mL) ( A ) or AGEs (advanced glycation end products) (10/100/200 µg/mL) (B , C ). Percentage of residual wound area (right panel) after treatment with HMGB1 (100 ng/mL) ( A ) or AGEs (10/100/200 µg/mL) ( B , C ), compared with 0 hours and standardized to the untreated condition (100%) (n = 5 experiments, each conducted in duplicate). ( D ) Representative images of scratch assays performed on HCE cells transiently transfected with siRNA against RAGE (siRNA RAGE) or siRNA control (Scramble) (100 nM) for 36 hours and then treated with AGEs (100 µg/mL) (left panel). Percentage of the residual wound area of HCE cells transfected with siRNA against RAGE (100 nM) for 36 hours and then treated with AGEs (100 µg/mL), compared with 0 h (right panel) (n = 5 experiments, each conducted in duplicate). Each bar graph shows mean ± SEM. Mann-Whitney test after a nonparametric ANOVA analysis; * P < 0.05; ** P < 0.01; *** P < 0.005; ns: not significant.

Article Snippet: Cells were transfected with 1 µg RAGE expression vector (RG204664; Origene, Herford, Germany) in the presence of 125 µL opti-MEM 1× and 5 µL p3000 reagent, mixed with 3.75 µL Lipofectamine 3000 reagent in 125 µL of opti-MEM (1×).

Techniques: Transfection, MANN-WHITNEY

Journal: Investigative Ophthalmology & Visual Science

Article Title: Advanced Glycation End Products and Receptor (RAGE) Promote Wound Healing of Human Corneal Epithelial Cells

doi: 10.1167/iovs.61.3.14

Figure Lengend Snippet: Functionality of the RAGE pathway. Characterization of the RAGE ( A ) mRNA and ( B ) protein expression in human cornea, primary human epithelial cells (mRNA only), and the HCE cell line (mRNA and protein) evaluated by ( A ) RT-PCR, ( B ) immunofluorescence, and ( B ) western blotting. For RT-PCR, negative controls (NC) were performed ( A ) without cDNA. ( B , left panel) Representative images of RAGE expression ( green ) in human corneas (top panel) and HCE cells (bottom panel). Nuclei were stained with Hoechst ( blue ); NC (left) were obtained by incubating HCE cells without primary antibody. ( B , right panel) Western blot experiments identified the RAGE protein at the described molecular weight (46 kDa). ( C ) Functionality of the NF-κB pathway (by luciferase reporter gene activity) was measured after treatment of HCE cells with AGEs (100 µg/mL) for 45 minutes (n = 5 experiments, each conducted in duplicate) (right panel). Positive controls (T+) were obtained by co-transfection with pMEKK (n = 5 experiments, each conducted in duplicate) (left panel). Each bar graph shows mean ± SEM. Mann-Whitney; * P < 0.05.

Article Snippet: Cells were transfected with 1 µg RAGE expression vector (RG204664; Origene, Herford, Germany) in the presence of 125 µL opti-MEM 1× and 5 µL p3000 reagent, mixed with 3.75 µL Lipofectamine 3000 reagent in 125 µL of opti-MEM (1×).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Western Blot, Staining, Molecular Weight, Luciferase, Activity Assay, Cotransfection, MANN-WHITNEY

Journal: Investigative Ophthalmology & Visual Science

Article Title: Advanced Glycation End Products and Receptor (RAGE) Promote Wound Healing of Human Corneal Epithelial Cells

doi: 10.1167/iovs.61.3.14

Figure Lengend Snippet: Cx43 expression is induced by the AGEs/RAGE axis in HCE cells. Characterization of Cx43 protein expression ( arrow ) by immunostaining (bottom panel). Cells incubated without primary antibody served as a negative control (NC). ( B ) Relative quantification of Cx43 mRNA expression in untreated HCE cells 0, 6, and 24 hours after scratch wounding. Results were expressed as a ratio of the 0-hour condition (n = 5 experiments, each conducted in duplicate). ( C ) Quantification of Cx43 mRNA expression in HCE cells treated with AGEs (100 µg/mL) for 6 hours or 24 hours without scratch wounding (left panel) or with scratch wounding (right panel). Cells not treated with AGEs served as a control. Results were expressed as a ratio of the untreated condition at each time point (n = 5 experiments, each conducted in duplicate). (Left panel) Quantification of Cx43 mRNA expression in HCE cells transiently transfected with siRNA against RAGE (siRNA RAGE) (100 nM) for 36 hours and then treated with AGEs (100 µg/mL) for 6 hours. Results were expressed as a ratio of the scrambled siRNA condition (n = 5 experiments, each conducted in duplicate). Each bar graph shows mean ± SEM. Mann-Whitney after a nonparametric ANOVA analysis; * P < 0.05; ns: not significant. ( D ) Relative quantification of Cx43 protein expression in HCE cells following treatment with AGEs (100 µg/mL) after scratch wounding. Untreated cells served as a control. Results were expressed as a ratio of the 0-hour, unwounded condition (n = 5 experiments) (top panel). Quantification of Cx43 protein expression in HCE cells treated with AGEs (100 µg/mL) for 12 and 24 hours after scratch wounding (bottom panel). Results were expressed as a ratio of the untreated condition at each time point (n = 5 experiments, each conducted in duplicate) (** P < 0.01). (E) Characterization by immunostaining of Cx43 protein expression according to the distance from the wound, time, and treatment with AGEs (100 µg/mL). Staining of Cx43 protein expression in HCE cells treated near the wound margins (left panel) and behind the wound (right panel). Cells not treated with AGEs (100 µg/mL) served as a control. Cells incubated without primary antibody served as a negative control (NC).

Article Snippet: Cells were transfected with 1 µg RAGE expression vector (RG204664; Origene, Herford, Germany) in the presence of 125 µL opti-MEM 1× and 5 µL p3000 reagent, mixed with 3.75 µL Lipofectamine 3000 reagent in 125 µL of opti-MEM (1×).

Techniques: Expressing, Immunostaining, Incubation, Negative Control, Transfection, MANN-WHITNEY, Staining

Journal: Scientific Reports

Article Title: Sestrin2 inhibits mTORC1 through modulation of GATOR complexes

doi: 10.1038/srep09502

Figure Lengend Snippet: (A and B) Sestrin2 destabilizes physical interaction between GATOR1 and GATOR2 in HEK293 cells. HA-tagged Sestrin2 was co-transfected with GATOR1 and GATOR2 components as indicated. NPRL2, a GATOR1 component, or WDR24, a GATOR2 component, was IPed using Flag antibody in (A) and (B), respectively. Input (WCL) and IP complex were analyzed by IB. (C) Purification of Sestrin2, GATOR1 and GATOR2 proteins. Sestrin2 and HA-GATOR1/2 complexes were purified from transformed E. coli and transfected HEK293 cells, respectively. (D) Sestrin2 inhibits assembly of GATOR1-GATOR2 supercomplex in vitro . As indicated, the purified proteins were mixed and incubated together. GATOR1 was pulled down from the mixture using NPRL2 antibody. GATOR1 and co-purified GATOR2 was detected through IB with HA antibody. (E) Sestrin2 overexpression decreases GATOR1-GATOR2 association in mouse liver. 10 9 pfu of adenoviruses expressing GFP or Sestrin2 were injected into two-month-old WT mice through the tail vein. After 4 days, liver lysates were prepared, and endogenous GATOR2 was IPed with WDR24 antibody. WCL and IP complex were analyzed by IB. (F) Sestrin2 is required for the effect of tunicamycin (Tm) on GATOR1-GATOR2 interaction. Two-month-old WT or Sesn2 −/− mice were injected with Tm (500 mg/Kg i.p.) as described . After 24 hrs, liver lysates were prepared, and GATOR2 was IPed with WDR24 antibody. WCL and IP complex were analyzed by IB. Cropped gel images are used in this figure and the gels were run under the same experimental conditions.

Article Snippet: Adenoviruses expressing human Sestrin2 (130233A) were purchased from Applied Biological Materials Inc. Epitope-tagged RagB/C and GATOR1/2 plasmids (19301, 19304, 46327-46333 and 46335; all in the pRK5 expression vector), which were originally generated in Dr. Sabatini's lab , were purchased from Addgene.

Techniques: Transfection, Purification, Transformation Assay, In Vitro, Incubation, Over Expression, Expressing, Injection

Journal: Scientific Reports

Article Title: Sestrin2 inhibits mTORC1 through modulation of GATOR complexes

doi: 10.1038/srep09502

Figure Lengend Snippet: (A) Inactivation of GST-RagB by Sestrin2. Flag-tagged Sestrin2 was co-transfected with GST-tagged RagB. Following metabolic labeling of the guanine nucleotide pools with 32 P, GST-RagB was pulled-down using Glutathione-Sepharose 4B beads. RagB-bound nucleotides were separated by thin layer chromatography (TLC) and visualized through autoradiography. The percentage of GDP-bound RagB protein is shown below each lane. Expression of Sestrin2 and GST-RagB were analyzed by IB of WCL. (B-D) Inactivation of endogenous RagB by Sestrin2-potentiated GATOR1. HEK293 cells were transduced with GFP (-) or Sestrin2-overexpressing (+) lentiviruses. After 48 h, RagB was IPed from cell lysates using RagB antibody (B,C) or NPRL2 antibody (D). 32 P-labeled nucleotides in the IP complex were separated by TLC and visualized through autoradiography (B). Input (WCL) and IP complex were analyzed by IB of endogenous proteins (C,D). (E) Sestrin2 promotes physical association between GATOR1 and RagB/RagC heterodimer. Sestrin2 was co-transfected with GATOR1, GATOR2 and GST-tagged RagB/RagC proteins as indicated. RagB/RagC proteins were pulled-down using Glutathione-Sepharose 4B beads. Input (WCL) and GST-purified complex (IP: GST) were analyzed by IB. Cropped gel images are used in this figure and the gels were run under the same experimental conditions.

Article Snippet: Adenoviruses expressing human Sestrin2 (130233A) were purchased from Applied Biological Materials Inc. Epitope-tagged RagB/C and GATOR1/2 plasmids (19301, 19304, 46327-46333 and 46335; all in the pRK5 expression vector), which were originally generated in Dr. Sabatini's lab , were purchased from Addgene.

Techniques: Transfection, Labeling, Thin Layer Chromatography, Autoradiography, Expressing, Transduction, Purification

Journal: Scientific Reports

Article Title: Sestrin2 inhibits mTORC1 through modulation of GATOR complexes

doi: 10.1038/srep09502

Figure Lengend Snippet: (A) Sestrin2 inhibits phosphorylation of mTORC1 targets through GATOR. HEK293 cells stably transduced with indicated shRNA were infected with GFP or Sestrin2-overexpressing lentiviruses. After 48 h, cells were analyzed by IB with indicated antibodies against endogenous proteins. (B and C) Silencing Npr2, the Drosophila homologue of NPRL2, relieves Drosophila Sestrin (dSesn)-induced growth arrest. Anterior (B, upper panels) and dorsal (B, lower panels) views of wing blades with ap-GAL4 –driven expression of indicated transgenic elements were imaged under a dissection microscope and a transmitted light microscope, respectively. Dorsal wing area were quantified and presented as a bar graph (C, n = 7, means ± s.e.m.). P value was calculated using Student's t-test. (D and E) Autophagy defects in dSesn -null animals were corrected by heterozygotic mutations of GATOR2 components. Thoraces from 5 day-old flies of indicated strains were analyzed through immunoblotting of dAtg8 and Actin proteins (D). Relative levels of unprocessed (dAtg8-I) and processed (dAtg8-II) dAtg8 proteins were quantified by densitometry and presented as a bar graph (E, n = 3, means ± s.e.m.). P values were calculated using Student's t-test. (F and G) Proposed model of the Sestrin2-GATOR-RagB pathway that mediates stress-induced mTORC1 silencing. In unstressed conditions, GATOR1 is constitutively inhibited by GATOR2, and RagB recruits mTORC1 to lysosomal surface and activates it as well as its downstream targets (F). Upon stress-induced expression of Sestrin2, GATOR1 is released from GATOR2-mediated inhibition and inactivates RagB (G). In the absence of active RagB, mTORC1 is released from lysosomal surface and mTORC1 signaling is subsequently inactivated. Cropped gel images are used in this figure and the gels were run under the same experimental conditions.

Article Snippet: Adenoviruses expressing human Sestrin2 (130233A) were purchased from Applied Biological Materials Inc. Epitope-tagged RagB/C and GATOR1/2 plasmids (19301, 19304, 46327-46333 and 46335; all in the pRK5 expression vector), which were originally generated in Dr. Sabatini's lab , were purchased from Addgene.

Techniques: Stable Transfection, Transduction, shRNA, Infection, Expressing, Transgenic Assay, Dissection, Microscopy, Light Microscopy, Western Blot, Inhibition