Journal: The Journal of Cell Biology

Article Title: β-Arrestin–Dependent Endocytosis of Proteinase-Activated Receptor 2 Is Required for Intracellular Targeting of Activated Erk1/2

doi:

Figure Lengend Snippet: PAR2-mediated activation of ERK1/2. (a) KNRK-PAR2+ARR-GFP cells (•) and KNRK-PAR2+ARR 319-418 -GFP cells (○) and hBRIE cells (□) were incubated with 50 nM trypsin for 0–60 min at 37°C, and ERK activity was measured using the MBP assay. (b–d) Western blots using antibodies to pERK1/2. (b) KNRK-PAR2+ARR-GFP cells (•), KNRK-PAR2 cells (⋄), and KNRK-PAR2+ARR 319-418 -GFP cells (○) were incubated with 50 nM trypsin. (c) hBRIE cells (□), hBRIE+ARR-GFP (⋄), and hBRIE+ARR 319-418 -GFP (♦) were incubated with 50 nM trypsin for 0–30 min at 37°C. (d) KNRK-PAR2+ARR-GFP cells (•) and KNRK-PAR2+ARR 319-418 -GFP cells (○) were incubated 50 μM AP for 0–30 min at 37°C. * P < 0.05 compared with cells expressing PAR2 alone or PAR2 plus ARR-GFP cells, n = 4.

Article Snippet: Kirsten murine sarcoma virus–transformed rat kidney epithelial cells (KNRK) were from the American Type Tissue Culture Collection (ATCC CRL 1569).

Techniques: Activation Assay, Incubation, Activity Assay, Western Blot, Expressing

Journal: The Journal of Cell Biology

Article Title: β-Arrestin–Dependent Endocytosis of Proteinase-Activated Receptor 2 Is Required for Intracellular Targeting of Activated Erk1/2

doi:

Figure Lengend Snippet: PAR2-mediated Ca 2+ mobilization in KNRK-PAR2 cells and KNRK-PAR2δ(ST363/6A) cells. (a) Concentration-response analysis for KNRK-PAR2 (•) and KNRK-PAR2 (δST363/6A) (▴) cells. (b–d) Each line shows [Ca 2+ ] i for individual KNRK-PAR2 cells (left) and KNRK-PAR2(δST363/6A) cells (right). (b) Note that the response to trypsin is prolonged in KNRK-PAR2(δST363/6A) cells. (c) Homologous desensitization in cells pretreated with 10 μM AP for 5 min before addition of 10 nM trypsin. (d) Heterologous desensitization in cells pretreated with 1 μM PDB for 20 min before addition of 10 nM trypsin. Note that KNRK-PAR2(δST363/6A) cells are resistant to desensitization.

Article Snippet: Kirsten murine sarcoma virus–transformed rat kidney epithelial cells (KNRK) were from the American Type Tissue Culture Collection (ATCC CRL 1569).

Techniques: Concentration Assay

Journal: The Journal of Cell Biology

Article Title: β-Arrestin–Dependent Endocytosis of Proteinase-Activated Receptor 2 Is Required for Intracellular Targeting of Activated Erk1/2

doi:

Figure Lengend Snippet: Agonist-induced PAR2 internalization. (a) Kinetics of PAR2 endocytosis in KNRK-PAR2+ARR-GFP cells (•), KNRK-PAR2 cells (⋄) KNRK-PAR2+ ARR 319-418 -GFP cells (○), KNRK-PAR2(δST363/6A) cells (▴), and hBRIE cells (□). Cells were incubated with 50 μM AP for 0–30 min at 37°C and endocytosis was determined by measuring surface Flag immunoreactivity by flow cytometry. * P < 0.05 compared with cells expressing PAR2 alone or PAR2 plus ARR-GFP cells, n = 3. (b–d) Localization of PAR2 and β-arrestin by immunofluorescence and confocal microscopy. KNRK-PAR2+ARR-GFP cells (b), KNRK-PAR2(δST363/6A)+ ARR-GFP cells (c), or KNRK-PAR2(δST363/6A) (d) were incubated with 50 nM trypsin for 0–30 min at 37°C. PAR2 was localized by immunofluorescence and β-arrestin was detected using GFP (b and c) or by immunofluorescence (d). The same cells are shown in each row and the images in the right panel are formed by superimposition of the images from the other two panels in the same row. Representative of two experiments. In KNRK-PAR2+ARR-GFP cells, note redistribution of β-arrestin to the plasma membrane at 5 min (arrowheads) and to endosomes at 30 min (arrows), where it colocalizes with PAR2. In KNRK-PAR2(δST363/6A)+ARR-GFP cells and in KNRK-PAR2(δST363/6A), note that PAR2 remains at the plasma membrane (arrowheads) and β-arrestin remains in the cytosol (arrows) with no colocalization. Bar, 10 μm.

Article Snippet: Kirsten murine sarcoma virus–transformed rat kidney epithelial cells (KNRK) were from the American Type Tissue Culture Collection (ATCC CRL 1569).

Techniques: Incubation, Flow Cytometry, Expressing, Immunofluorescence, Confocal Microscopy, Clinical Proteomics, Membrane

Journal: The Journal of Cell Biology

Article Title: β-Arrestin–Dependent Endocytosis of Proteinase-Activated Receptor 2 Is Required for Intracellular Targeting of Activated Erk1/2

doi:

Figure Lengend Snippet: (a) PAR2-mediated ERK1/2 activity. KNRK-PAR2 (⋄) and KNRK-PAR2(δST363/6A) (▴) cells were treated with 50 nM trypsin for 0–30 min, and ERK activity was measured using the MBP assay. (b) Activation of ERK1/2. KNRK-PAR2(δST363/6A) (▴), KNRK-PAR2 (δST363/6A)+ARR-GFP (▵), and KNRK-PAR2 (δST363/6A)+ARR 319-418 -GFP (♦) cells were treated with 50 nM trypsin for 0–30 min and phosphorylation was assessed using antibodies to pERK1/2. * P < 0.05 compared with KNRK-PAR2 cells, n = 3.

Article Snippet: Kirsten murine sarcoma virus–transformed rat kidney epithelial cells (KNRK) were from the American Type Tissue Culture Collection (ATCC CRL 1569).

Techniques: Activity Assay, Activation Assay, Phospho-proteomics

Journal: The Journal of Cell Biology

Article Title: β-Arrestin–Dependent Endocytosis of Proteinase-Activated Receptor 2 Is Required for Intracellular Targeting of Activated Erk1/2

doi:

Figure Lengend Snippet: Mechanism of PAR2-mediated activation of ERK1/2. (a) KNRK-PAR2, hBRIE, KNRK-PAR2+ARR 319-418 , and KNRK-PAR2(δST363/6A) cells were untreated (control, con), or incubated with 100 nM GF109203X (GFX), 20 nM LY379196 (LY), 100 ng/ml PTX, 10 μM genistein (GEN), 20 μM tyrphostin 25 (TP), or were cotransfected with N17ras. Cells were incubated with 50 nM trypsin for 5 min. * P < 0.05 compared with untreated cells, n = 4. (b–f) Analysis of KNRK-PAR2 and KNRK-PAR2(δST363/6A) cells by immunoprecipitation (IP) and Western blotting (WB). Cells were incubated with 50 nM trypsin for 5 min. Extracts were immunoprecipitated with antibodies to PYK2 (b), Shc (c and d), and src (e–g). Blots were probed for phosphotyrosine (b–e), PYK2 (f), and src (g).

Article Snippet: Kirsten murine sarcoma virus–transformed rat kidney epithelial cells (KNRK) were from the American Type Tissue Culture Collection (ATCC CRL 1569).

Techniques: Activation Assay, Control, Incubation, Immunoprecipitation, Western Blot

Journal: The Journal of Cell Biology

Article Title: β-Arrestin–Dependent Endocytosis of Proteinase-Activated Receptor 2 Is Required for Intracellular Targeting of Activated Erk1/2

doi:

Figure Lengend Snippet: Nuclear translocation of ERK1/2. (a–d) Subcellular fractionation of activated ERK1/2. KNRK-PAR2+ARR-GFP cells (•), KNRK-PAR2+ ARR 319-418 -GFP cells (○), or PAR2(δST363/6A) cells (s) were incubated with 50 nM trypsin for 0–30 min at 37°C, and pERK was determined in the cytosolic (a) and nuclear (b) fractions ( n = 3). hBRIE cells were incubated with 50 nM trypsin (□) or 10% serum (♦), and pERK was determined in the cytosolic (c) and nuclear (d) fractions. (e) KNRK-PAR2 and KNRK-PAR2(δST363/6A) cells, transiently transfected with ERK2-GFP, were incubated with 50 nM trypsin at 37°C, and translocation of ERK2-GFP was observed by confocal imaging. Representative of eight experiments. In KNRK-PAR2 cells, note that ERK2-GFP remains cytosolic but, in KNRK-PAR2(δST363/6A) cells, it redistributes to the nucleus. (f and g) Proliferative responses to AP and serum. KNRK-PAR2 (f, □) and KNRK-PAR2(δST363/6A) cells (f, ▪) or hBRIE cells (g) were incubated with 50 μM AP or 20% FCS for 24 h, and incorporation of [ 3 H]thymidine and cell number were measured. * P < 0.05 compared with untreated cells or KNRK-PAR2 cells, n = 3.

Article Snippet: Kirsten murine sarcoma virus–transformed rat kidney epithelial cells (KNRK) were from the American Type Tissue Culture Collection (ATCC CRL 1569).

Techniques: Translocation Assay, Fractionation, Incubation, Transfection, Imaging

Journal: The Journal of Cell Biology

Article Title: β-Arrestin–Dependent Endocytosis of Proteinase-Activated Receptor 2 Is Required for Intracellular Targeting of Activated Erk1/2

doi:

Figure Lengend Snippet: Trypsin induced association of β-arrestin and raf-1. (a and b) Localization of β-arrestin and raf-1 by immunofluorescence and confocal microscopy. KNRK-PAR2+ARR-GFP cells (a) or KNRK-PAR2(δST363/6A)+ ARR-GFP cells (b) were incubated with 50 nM trypsin for 0 or 5 min at 37°C. β-Arrestin was localized using GFP and raf-1 was localized by immunofluorescence. The same cells are shown in each row and the images in the right panel are formed by superimposition of images from the other two panels in the same row. Representative of two experiments. In KNRK-PAR2+ARR-GFP cells, note the redistribution of β-arrestin and raf-1 from the cytosol at 0 min (arrows) to the plasma membrane at 5 min (arrowheads), where they colocalize. In KNRK-PAR2 (δST363/6A)+ARR-GFP cells, note that β-arrestins remain in the cytosol (arrows) and raf-1 redistributes to the plasma membrane at 5 min (arrowheads). (c–f) Coimmunoprecipitation of raf-1 and β-arrestin. Cells were incubated with 50 nM trypsin for 0–30 min at 37°C, lysed, immunoprecipitated (IP) using antibodies to GFP or β-arrestin-1/2, and analyzed by Western blotting (WB) with a raf-1 antibody. (c) In KNRK-PAR2 cells, but not in KNRK-PAR2(δST363/6A) cells, β-arrestin and raf-1 coprecipitated. (d) Similarly, in KNRK-PAR2+ARR-GFP cells but not KNRK-PAR2(δST363/6A)+ARR-GFP cells ARR-GFP and raf-1 coprecipitated with antibodies to GFP. (e) In KNRK-PAR2+ARR 319-418 -GFP cells, endogenous β-arrestin and raf-1 coprecipitated, but ARR 319-418 and raf-1 did not coprecipitate. (f) In hBRIE+ARR-GFP cells, ARR-GFP and raf-1 coprecipitated. Bars, 10 μm.

Article Snippet: Kirsten murine sarcoma virus–transformed rat kidney epithelial cells (KNRK) were from the American Type Tissue Culture Collection (ATCC CRL 1569).

Techniques: Immunofluorescence, Confocal Microscopy, Incubation, Clinical Proteomics, Membrane, Immunoprecipitation, Western Blot

Journal: The Journal of Cell Biology

Article Title: β-Arrestin–Dependent Endocytosis of Proteinase-Activated Receptor 2 Is Required for Intracellular Targeting of Activated Erk1/2

doi:

Figure Lengend Snippet: Gel filtration analysis of an ERK signaling complex. KNRK-PAR2 (a and b), KNRK-PAR2(δST363/6A) cells (c and d), or hBRIE cells (e and f), or KNRK-PAR2+ARR 319-418 -GFP cells (g) were untreated (a, c, and e) or incubated with 50 μM AP (b, d, f, and g) for 5 min. Cell lysates were fractionated on a S300 Sephacryl column. The presence of pERK, raf-1, β-arrestin-1, and PAR2 in each fraction was determined by Western blotting (inset). PAR2 was detected using HA.11 antibody in KNRK cells and 2N antibody in hBRIE cells. Results are expressed as a percentage of the total protein for each partition coefficient (σ) ( n = 3). The bracketed columns represent regions where proteins coeluted. Representative Western blots are shown of fractions containing the complex in KNRK-PAR2 cells (★, σ = 0.34), hBRIE cells (⋆, σ = 0.31–34), and in KNRK-PAR2+ARR 319-418 -GFP (*, σ = 0.45). (h) The Stoke's radii of the four molecular mass standards and the complex in KNRK-PAR2 cells (★, ∼6.2), hBRIE cells (⋆, ∼6.2–6.6), and KNRK-PAR2+ARR 319-418 -GFP (*, ∼5 nm) are graphed as a function of the error function complement of σ.

Article Snippet: Kirsten murine sarcoma virus–transformed rat kidney epithelial cells (KNRK) were from the American Type Tissue Culture Collection (ATCC CRL 1569).

Techniques: Filtration, Incubation, Western Blot

Journal: The Journal of Cell Biology

Article Title: β-Arrestin–Dependent Endocytosis of Proteinase-Activated Receptor 2 Is Required for Intracellular Targeting of Activated Erk1/2

doi:

Figure Lengend Snippet: Coprecipitation of components of the β-arrestin–containing signaling complex. (a) Fractions from the gel filtration columns of AP-stimulated KNRK-PAR2 cells at partition coefficients 0.31–0.34 were pooled, concentrated, and immunoprecipitated with pERK or β-arrestin-1/2 antibodies. Western blots were probed for PAR2 using the HA.11 antibody, β-arrestin-1, raf-1, and pERK. (b) Fractions from the gel filtration columns of AP-stimulated hBRIE cells at partition coefficients 0.31–0.34 were similarly processed, immunoprecipitated with a β-arrestin-1/2 antibody, and blotted with antibodies to pERK and raf-1.

Article Snippet: Kirsten murine sarcoma virus–transformed rat kidney epithelial cells (KNRK) were from the American Type Tissue Culture Collection (ATCC CRL 1569).

Techniques: Filtration, Immunoprecipitation, Western Blot

Journal: Oncotarget

Article Title: A novel pan-Nox inhibitor, APX-115, protects kidney injury in streptozotocin-induced diabetic mice: possible role of peroxisomal and mitochondrial biogenesis

doi: 10.18632/oncotarget.18540

Figure Lengend Snippet: Diabetes was induced in mice by intraperitoneal injection of STZ (50 mg/kg). Then APX-115 (60 mg/kg/day) or losartan (1.5 mg/kg/day) was administered orally for 12 weeks to diabetic mice. After 12 weeks, urine and blood samples were collected for analysis of ( A ) urinary albumin excretion, ( B ) albumin/creatinine ratio, ( C ) creatinine clearance rate, and ( D ) plasma cystatin C. ( E ) Kidneys were fixed in paraffin and cut into 3 μm sections that were subsequently stained with PAS reagent. Scale bar: 10 μm; original magnification: 630×. After PAS staining, ( F ) glomerular volume, ( G ) mesangial area, and ( H ) tuft area were analyzed using Image-Pro Plus 4.5.1. DM, STZ-induced diabetic mice. Data are presented as means ± SE of 10–12 mice/group; * p < 0.05 vs. control, † p < 0.05 vs. DM.

Article Snippet: A murine mesangial cell line (MES-13, cloned from mice transgenic for the early region of SV-40 virus, passage 25) was obtained from American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Injection, Clinical Proteomics, Staining, Control

Journal: Oncotarget

Article Title: A novel pan-Nox inhibitor, APX-115, protects kidney injury in streptozotocin-induced diabetic mice: possible role of peroxisomal and mitochondrial biogenesis

doi: 10.18632/oncotarget.18540

Figure Lengend Snippet: ( A ) Plasma LPO, ( B ) urinary LPO, ( C ) kidney tissue LPO, ( D ) Nox1, ( E ) Nox2, and ( F ) Nox4 mRNA expression levels in kidneys were measured using real-time PCR. ( G and H ) Frozen kidney sections were stained with DHE at 5 µM (original magnification: 400×; scale bar: 20 μm). (A–H) Data are presented as means ± SE of 10–12 mice/group; * p < 0.05 vs. control, † p < 0.05 vs. DM. ( I ) Mesangial cells were incubated with or without APX-115 (1 µM) for 30 min and stimulated with or without 30 mM high glucose (HG) for 24 h followed by angII for 30 min. After that cells were incubated with 10 µM DCF-DA for 10 min and the fluorescence intensity was measured with a Zeiss vision system. Data are presented as means ± SE of at least 2 independent experiments; * p < 0.05 vs. control, † p < 0.05 vs. angII or angII+HG in DMSO.

Article Snippet: A murine mesangial cell line (MES-13, cloned from mice transgenic for the early region of SV-40 virus, passage 25) was obtained from American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Clinical Proteomics, Expressing, Real-time Polymerase Chain Reaction, Staining, Control, Incubation, Fluorescence