Journal: International Journal of Dentistry

Article Title: Acinar Cell Proliferation Promoted by BMP2 in Injured Mouse Parotid Gland: BMP2 Promotes Cell Proliferation in Parotid Gland

doi: 10.1155/2023/1765317

Figure Lengend Snippet: Mouse parotid acinar cells in primary culture. (a) Cell proliferative capacity of BMP2-added (100 ng/mL) and nonadded (control) groups after 48 hr of culture. The proliferative capacity of BMP2-added cultured cells was significantly increased compared to that in the control group. ∗∗ Indicates significance at P < 0.01, ∗ P < 0.05. Data were shown as mean ± SD. Three independent experiments were performed. (b) Protein expression of E-cadherin (an epithelial marker) and vimentin (a mesenchymal marker) in cultured cells of each group at 48 hr after the addition of BMP2. NIH3T3 (3T3) was used as a positive control for mesenchymal markers. RS: rat serum.

Article Snippet: NIH3T3 whole cell lysate (Novus Biologicals, Centennial, CO, USA) was used as a positive control for mesenchymal markers.

Techniques: Control, Cell Culture, Expressing, Marker, Positive Control

Journal: The Journal of Biological Chemistry

Article Title: An Advanced Glycation End Product (AGE)-Receptor for AGEs (RAGE) Axis Restores Adipogenic Potential of Senescent Preadipocytes through Modulation of p53 Protein Function *

doi: 10.1074/jbc.M112.399790

Figure Lengend Snippet: Replicative senescence-impaired adipogenic potential of 3T3-L1 preadipocytes and a positive correlation of AGE-RAGE axis with adipose tissue aging and adiposity. A, ORO staining of differentiated 3T3-L1 preadipocytes (Pre) with different passage numbers (e.g. p.5, p.10, and p.15) for 8 days. B, gene expression analysis of adiponectin and PPARγ in differentiated preadipocytes with passage number of ≤5 (LP) and ≥15 (HP) by real time PCR. C, SA-β-galactosidase activity assay in LP and HP 3T3-L1 preadipocytes as shown in the overall view (top panel) and microscopic image (middle panel, ×100 magnification) and quantification of SA-β-gal-positive cells (bottom panel). D, real time PCR analysis of mRNA levels of p53 and IL-6 in LP and HP preadipocytes. E, levels of ROS in LP and HP preadipocytes were detected by DCHF-DA assay. The fluorescent signals in these cells were viewed by fluorescence microscopy (top panel, ×100 magnification) and quantified by a fluorescence microplate reader (bottom panel). F, LP and HP 3T3-L1 preadipocytes were treated with or without 167 nm insulin for 30 or 60 min. The levels of phosphorylated Akt (p-Akt(Ser473)), total Akt, and β-actin in these cells were analyzed by immunoblot assay. G, RT-PCR analysis of RAGE, adiponectin, and PPARγ in preadipocytes (D0) and differentiated adipocytes (D9) of LP and HP 3T3-L1 cells. H, immunoblot analysis of inguinal fat pad isolated from 6-week-old and 1-year-old chow diet-fed male mice (left panel), and quantification of intensity of the major CML-modified protein (∼55 kDa) and RAGE and p53 bands (right panel). I, immunoblot analysis of epididymal fat pads isolated from 14-week-old chow diet-fed lean and high fat diet-fed obese male mice (left panel) and quantification of intensity of the major CML modified protein (∼55 kDa) and RAGE and aP2 bands (right panel). J, real time PCR analysis of RAGE in epididymal fat pads isolated from lean and obese mice. Bars represent mean ± S.E. (n = 3–5). *, p < 0.05; **, p < 0.01; ***, p < 0.001. All experiments were repeated at least 2–3 times.

Article Snippet: In a RAGE neutralization study, 10 μg/ml anti-RAGE antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) was added to the differentiating 3T3-L1 cells from day 0 to day 2.

Techniques: Staining, Expressing, Real-time Polymerase Chain Reaction, Activity Assay, Fluorescence, Microscopy, Western Blot, Reverse Transcription Polymerase Chain Reaction, Isolation, Modification

Journal: The Journal of Biological Chemistry

Article Title: An Advanced Glycation End Product (AGE)-Receptor for AGEs (RAGE) Axis Restores Adipogenic Potential of Senescent Preadipocytes through Modulation of p53 Protein Function *

doi: 10.1074/jbc.M112.399790

Figure Lengend Snippet: Pretreatment of glycated BSA leads to an induction of adipogenesis in senescent HP 3T3-L1 preadipocytes, MEF, and inguinal SV cells. A, schematic of glycated BSA (MG- and GA-BSA, 300 μg/ml) pretreatment of HP 3T3-L1 preadipocytes prior to an 8-day adipogenesis (top panel). Representative ORO-stained image (middle panel) and quantification of ORO-stained HP adipocytes pretreated with BSA or glycated BSA (bottom panel) are shown. Representative microscopic image of ORO-stained LP (passage number 2) and HP (passage number 7) MEF cells (B) and primary SV cells isolated from inguinal fat pads of 12-week-old and 1-year-old chow diet-fed male mice (C) were pretreated with BSA or glycated BSA prior to induction of adipogenesis. Cells were then stained with ORO and subjected to microscopic imaging (×100 magnification). D, RT-PCR analysis of differentiated inguinal SV cells in C. Bars represent mean ± S.E. (n = 3). **, p < 0.01; ***, p < 0.001.

Article Snippet: In a RAGE neutralization study, 10 μg/ml anti-RAGE antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) was added to the differentiating 3T3-L1 cells from day 0 to day 2.

Techniques: Staining, Isolation, Imaging, Reverse Transcription Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: An Advanced Glycation End Product (AGE)-Receptor for AGEs (RAGE) Axis Restores Adipogenic Potential of Senescent Preadipocytes through Modulation of p53 Protein Function *

doi: 10.1074/jbc.M112.399790

Figure Lengend Snippet: Binding between AGE-activated RAGE and p53 inhibits p53 expression in senescent preadipocytes. LP 3T3-L1 preadipocytes, control (sh-Control), and RAGE-knockdown (sh-RAGE) HP preadipocytes treated with or without 300 μg/ml BSA or MG-BSA/GA-BSA for 48 h were subjected to immunoblot analysis of RAGE, phosphorylated p53 (p-p53 (Ser15)) and p21 (A), and gene expression analysis of p53 and p21 (B). ChIP assay (C) and immunoprecipitation (D) of the HP preadipocytes used in A. IP, immunoprecipitation. IB, immunoblot. E, confluent HP preadipocytes were treated with pifithrin-α (PFT-α) at 0, 10, and 50 μm for 24 h followed by an exposure to 300 μg/ml BSA or MG-BSA/GA-BSA for additional 48 h in the absence of pifithrin-α. These cells were subjected to adipogenesis for 8 days. Lipid accumulation in differentiated HP preadipocytes was detected by ORO staining (left panel) and a quantitative ORO staining analysis (right panel). Bars represent mean ± S.E. (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001. N.S., not significant. ##, p < 0.01 and ###, p < 0.001 versus BSA. All experiments were repeated at least 2–3 times.

Article Snippet: In a RAGE neutralization study, 10 μg/ml anti-RAGE antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) was added to the differentiating 3T3-L1 cells from day 0 to day 2.

Techniques: Binding Assay, Expressing, Western Blot, Immunoprecipitation, Staining

Journal: The Journal of Biological Chemistry

Article Title: An Advanced Glycation End Product (AGE)-Receptor for AGEs (RAGE) Axis Restores Adipogenic Potential of Senescent Preadipocytes through Modulation of p53 Protein Function *

doi: 10.1074/jbc.M112.399790

Figure Lengend Snippet: Glycated BSA promotes adipogenesis of senescent preadipocytes. A, schematic of BSA or glycated BSA (MG- and GA-BSA, 300 μg/ml) treatment of LP and HP 3T3-L1 preadipocytes during an 8-day of adipogenesis (top panel). Representative ORO-stained images of these cells (bottom panel). B, quantification of ORO-stained 3T3-L1 cells in A. Real time PCR analysis (C) and RT-PCR analysis (D) of genes in HP preadipocytes differentiated in the presence or absence of 300 μg/ml BSA or glycated BSA. Bars represent mean ± S.E. (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001. All experiments were repeated at least 2–3 times.

Article Snippet: In a RAGE neutralization study, 10 μg/ml anti-RAGE antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) was added to the differentiating 3T3-L1 cells from day 0 to day 2.

Techniques: Staining, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: An Advanced Glycation End Product (AGE)-Receptor for AGEs (RAGE) Axis Restores Adipogenic Potential of Senescent Preadipocytes through Modulation of p53 Protein Function *

doi: 10.1074/jbc.M112.399790

Figure Lengend Snippet: RAGE is required for AGE-induced adipogenesis in senescent preadipocytes. A, immunoblot analysis (top left panel) and quantification (bottom left panel) of RAGE in LP and HP 3T3-L1 preadipocytes exposed to BSA (300 μg/ml) or MG-BSA or GA-BSA (300 μg/ml) for 48 h. Immunofluorescent images of RAGE (green) and DAPI-stained nucleus (blue) of HP 3T3-L1 preadipocytes treated with or without 300 μg/ml BSA, MG-BSA, or GA-BSA for 48 h (right panel). Scale bars, 20 μm. B, ORO staining (left panel) and quantitative ORO staining analyses (right panel) of HP 3T3-L1 preadipocytes differentiated for 8 days after pretreatment with 300 μg/ml BSA or glycated BSA from day −2 to day 0 in the presence or absence of 10 μg/ml anti-RAGE antibody. C, HP 3T3-L1 preadipocytes infected with control lentivirus (sh-Control) or lentivirus containing RAGE-specific shRNA (sh-RAGE) for 24 h were subjected to RT-PCR analysis of RAGE mRNA (left panel). Immunoblot analysis of RAGE in lentivirus-infected HP 3T3-L1 preadipocytes treated with 300 μg/ml BSA, MG-BSA, or GA-BSA for 48 h (right panel). Lentivirus-infected HP preadipocytes were pretreated with 300 μg/ml BSA, MG-BSA, or GA-BSA for 48 h followed by an 8-day adipogenesis. These cells were subjected to ORO staining analysis (D) and real time PCR analysis of RAGE, PPARγ, and FAS (E). Pre, preadipocytes; Adipo, adipocytes; N.S., not significant. Bars represent mean ± S.E. (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001. #, p < 0.05 versus BSA. All experiments were repeated at least 2–3 times.

Article Snippet: In a RAGE neutralization study, 10 μg/ml anti-RAGE antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) was added to the differentiating 3T3-L1 cells from day 0 to day 2.

Techniques: Western Blot, Staining, Infection, shRNA, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Journal: Journal of Lipid Research

Article Title: Dopamine D2 receptor upregulates leptin and IL-6 in adipocytes [S]

doi: 10.1194/jlr.M081000

Figure Lengend Snippet: D2R and D3R are expressed in human adipocytes and mouse adipocytes. A, B: The expression of D2R and D3R was determined by Western blot in human subcutaneous adipocytes cells and 3T3-L1 cells. Mouse proximal tubular cells were used as a positive control (PC). PA, preadipocites; A, adipocytes. C:. D2R and D3R mRNA expression was determined by qRT-PCR in human preadipocytes and adipocytes. The data were normalized with GAPDH.

Article Snippet: 3T3-L1 preadipocyte medium (PM-1-L1), 3T3-L1 differentiation medium (DM-2-L1), and 3T3-L1 adipocyte medium (AM-1-L1) were from Zen-Bio, Inc. Differentiated 3T3-L1 adipocyte cells were serum-starved for 2 h and treated for 24 h with 1 μM quinpirole (D 2 R/D 3 R agonist, Sigma-Aldrich), or 1 μM quinpirole plus 1 μM L-741,262 (selective D 2 R antagonist, Sigma-Aldrich), as previously described ( 15 , 16 ).

Techniques: Expressing, Western Blot, Positive Control, Quantitative RT-PCR

Journal: Journal of Lipid Research

Article Title: Dopamine D2 receptor upregulates leptin and IL-6 in adipocytes [S]

doi: 10.1194/jlr.M081000

Figure Lengend Snippet: D2R stimulation increases leptin, IL-6, and TNF α expression in 3T3-L1 cells. A: 3T3-L1 cells were treated with vehicle or quinpirole (1 μM, 24 h), a D2R agonist, in the presence or absence of a D2R antagonist (L-7431,626, 1 μM, 24 h). The expression of adiponectin, visfatin, leptin, and IL-6 was determined by Western blot and normalized with GAPDH expression. Data are expressed as mean ± SEM, one-way ANOVA or Student’s t-test; *P < 0.05; n = 4. B: 3T3-L1 cells were treated with vehicle or quinpirole (1 μM, 15 min, 30 min, or 2 h). The expression of leptin and TNFα was determined by Western blot and normalized with GAPDH expression. Data are expressed as mean ± SEM, one-way ANOVA or Student’s t-test; *P < 0.05; n = 4. C: 3T3-L1 cells were treated with vehicle or quinpirole, a D2R agonist (1μM, 24 h). The mRNA expression of leptin, IL-6, TNFα, NFKB, and MCP-1 were determined by qRT-PCR and normalized with GAPDH. Data are expressed as mean ± SEM, Student’s t-test; *P < 0.05; n = 5. D: Concentration of leptin in the medium was determined by ELISA. Data are expressed as mean ± SEM, Student’s t-test; *P < 0.05; n = 3.

Article Snippet: 3T3-L1 preadipocyte medium (PM-1-L1), 3T3-L1 differentiation medium (DM-2-L1), and 3T3-L1 adipocyte medium (AM-1-L1) were from Zen-Bio, Inc. Differentiated 3T3-L1 adipocyte cells were serum-starved for 2 h and treated for 24 h with 1 μM quinpirole (D 2 R/D 3 R agonist, Sigma-Aldrich), or 1 μM quinpirole plus 1 μM L-741,262 (selective D 2 R antagonist, Sigma-Aldrich), as previously described ( 15 , 16 ).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Lipid Research

Article Title: Dopamine D2 receptor upregulates leptin and IL-6 in adipocytes [S]

doi: 10.1194/jlr.M081000

Figure Lengend Snippet: Downregulation of D2R decreases leptin, IL-6 in 3T3-L1 cells. 3T3-L1 cells were transfected with D2R siRNA (30 nM, 72 h) or mock (non-silencing) siRNA. A: The expression of D2R, D3R, D4R, leptin, and IL-6 was determined by Western blot and normalized by GAPDH. Data are expressed as mean ± SEM, Student’s t-test; *P < 0.05; n = 5/3. B: The concentration of leptin in the medium was determined by ELISA. Data are expressed as mean ± SEM, Student’s t-test; *P < 0.05; n = 5/3. C: The expression of leptin, IL-6, and TNFα was determined by qRT-PCR and normalized by GAPDH. Student’s t-test; *P < 0.05; n = 5.

Article Snippet: 3T3-L1 preadipocyte medium (PM-1-L1), 3T3-L1 differentiation medium (DM-2-L1), and 3T3-L1 adipocyte medium (AM-1-L1) were from Zen-Bio, Inc. Differentiated 3T3-L1 adipocyte cells were serum-starved for 2 h and treated for 24 h with 1 μM quinpirole (D 2 R/D 3 R agonist, Sigma-Aldrich), or 1 μM quinpirole plus 1 μM L-741,262 (selective D 2 R antagonist, Sigma-Aldrich), as previously described ( 15 , 16 ).

Techniques: Transfection, Expressing, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Journal of Lipid Research

Article Title: Dopamine D2 receptor upregulates leptin and IL-6 in adipocytes [S]

doi: 10.1194/jlr.M081000

Figure Lengend Snippet: PI3K inhibition blunts the stimulatory effect of D2R on leptin expression in 3T3-L1 cells. A: 3T3-L1 cells were treated with vehicle or quinpirole (1 μM; 24 h) in the presence or absence of LY294002 (10 μM; 24 h), an inhibitor of PI3K. The expression of phosphorylated-AKT, AKT, leptin, IL-6, and GAPDH was determined by Western blot; Student’s t-test or ANOVA; *P < 0.05, n = 4. B: The expression of OB-R was determined by Western blot in 3T3-L1 preadipocytes and 3T3-L1 adipocytes treated with quinpirole (1 μM; 24 h). C: 3T3-L1 adipocytes treated with L39A/D40A/F41 (25 nM, 24 h), a leptin antagonist, and quinpirole (1 μM; 24 h). The expression of IL-6 was determined by Western blot and normalized by GAPDH. Student’s t-test or ANOVA; *P < 0.05; n = 4.

Article Snippet: 3T3-L1 preadipocyte medium (PM-1-L1), 3T3-L1 differentiation medium (DM-2-L1), and 3T3-L1 adipocyte medium (AM-1-L1) were from Zen-Bio, Inc. Differentiated 3T3-L1 adipocyte cells were serum-starved for 2 h and treated for 24 h with 1 μM quinpirole (D 2 R/D 3 R agonist, Sigma-Aldrich), or 1 μM quinpirole plus 1 μM L-741,262 (selective D 2 R antagonist, Sigma-Aldrich), as previously described ( 15 , 16 ).

Techniques: Inhibition, Expressing, Western Blot

Journal: Journal of Lipid Research

Article Title: Dopamine D2 receptor upregulates leptin and IL-6 in adipocytes [S]

doi: 10.1194/jlr.M081000

Figure Lengend Snippet: Dopamine decreases leptin expression via adrenergic receptors. 3T3-L1 cells were treated with vehicle or dopamine (1 μM, 24 h), an antagonist of D1R (SCH 23390, 10 µM) and D2R (L741,626, 10 µM), α-adrenergic receptor antagonist (propranolol, 10 µM), and β-adrenergic receptor antagonist (phentolamine, 10 µM) in medium with or without insulin. Protein expression of leptin in the medium was determined by ELISA and normalized by protein concentration, one-way ANOVA; *P < 0.05, control vs. others; #P < 0.05, vs. others; Student’s t-test; †P < 0.05 vs. control, n = 6/11.

Article Snippet: 3T3-L1 preadipocyte medium (PM-1-L1), 3T3-L1 differentiation medium (DM-2-L1), and 3T3-L1 adipocyte medium (AM-1-L1) were from Zen-Bio, Inc. Differentiated 3T3-L1 adipocyte cells were serum-starved for 2 h and treated for 24 h with 1 μM quinpirole (D 2 R/D 3 R agonist, Sigma-Aldrich), or 1 μM quinpirole plus 1 μM L-741,262 (selective D 2 R antagonist, Sigma-Aldrich), as previously described ( 15 , 16 ).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Protein Concentration

Journal: Translational Oncology

Article Title: Prognostic Significance and Functional Role of CEP57 in Prostate Cancer 1

doi: 10.1016/j.tranon.2015.11.004

Figure Lengend Snippet: CEP57 overexpression induces microtubule bundling and promotes centriole overduplication. (A) Co-immunofluorescence microscopic analysis of CEP57 and α-tubulin to visualize CEP57-induced microtubule bundles (“basket”) in mouse NIH3T3 cells overexpressing either empty vector (control) or murine (mCEP57). Nuclei stained with DAPI. Scale bar = 10 μm. (B) Immunofluorescence microscopic analysis of centrioles in LNCaP cells expressing centrin-GFP as a centriole marker and transfected with either empty vector (control) or mCEP57. A DsRed encoding plasmid was used as transfection control. Nuclei stained with DAPI. Scale bar = 10 μm. (C) Quantification of overduplicated centrioles (> 4 centrioles per cell, > 1 daughter at a single maternal centriole) in LNCaP cells transfected with centrin-GFP together with an empty vector (control) or mCEP57. Statistical analysis was performed using Student's two-tailed t test for independent samples.

Article Snippet: Human prostate cancer cell lines LNCaP and PC-3 and mouse NIH3T3 cells were obtained from CLS Cell Line Service (Eppelheim, Germany) and maintained according to the distributor's recommendations.

Techniques: Over Expression, Immunofluorescence, Plasmid Preparation, Staining, Expressing, Marker, Transfection, Two Tailed Test

Journal: Journal of Biological Chemistry

Article Title: Src Family Kinases Mediate Epithelial Na+ Channel Inhibition by Endothelin

doi: 10.1074/jbc.m106919200

Figure Lengend Snippet: FIG. 1. Endothelin-1 potently inhib- its amiloride-sensitive currents. A, raw traces from IV curves generated from whole cell recordings in the presence and absence of 10 M amiloride confirm the expression and activity of ENaC in NIH 3T3 cells infected with ENaC genes. The amiloride-sensitive component (Œ Iamil) of the whole cell currents is calcu- lated from the difference in current before (f) and after (G) treatment with amilo- ride. B, treatment of NIH 3T3 cells with 10 nM ET-1 (G) reduces Iamil significantly below control (f). Cells were studied in the presence (f) or absence (G) of 10 nM ET-1 for 3–8 min. Results are average currents S.E. for six experiments under each condition. C, ET-1 inhibition of ENaC is dose-dependent. Cells were treated with 1 pM, 100 pM, or 10 nM ET-1 as in B, and full IV curves were gener- ated. Shown is Iamil for ET-1-treated cells expressed as a percentage of the control (no treatment) amiloride-sensitive cur- rent at 120 mV. Results represent the average currents S.E. for four to six experiments. Statistics were performed using Student’s t test (**, p 0.005; ***, p 0.0005).

Article Snippet: In Vitro Kinase Assays—Kinases were immunoprecipitated from NIH 3T3 cell lysates using an antibody directed against the conserved COOH terminus of the Src family kinase sequence that recognizes all Src kinase family members (rabbit polyclonal antibody; Santa Cruz), or using antibodies to specific Src family kinases ( -Src kinase, Oncogene; -Yes kinase, Wako).

Techniques: Inhibition, Generated, Expressing, Activity Assay, Infection, Control

Journal: Journal of Biological Chemistry

Article Title: Src Family Kinases Mediate Epithelial Na+ Channel Inhibition by Endothelin

doi: 10.1074/jbc.m106919200

Figure Lengend Snippet: FIG. 3. Src family kinase inhibitors prevent ET-1 inhibition of ENaC. A, in vitro kinase assays demonstrate the efficacy of PP2, a specific Src family kinase inhibitor. Purified, active Src kinase was incubated with a Src substrate, enolase, and [-32P]ATP in a modified pipette solution buffer. Under control conditions, Src kinase phosphorylated enolase (lane 1). In the presence of 1 M PP2, Src phosphorylation of enolase was inhibited (lane 2). Incubation with 1 M PP3, an inactive analogue of PP2, did not interfere with Src phosphorylation of enolase (lane 3). Lanes 4 and 5 are reactions performed without enolase or Src kinase, respectively. Results are representative of three separate experiments. B, whole cell currents were recorded from cells treated with 100 pM ET-1 alone (f) or following pretreatment with 1 M PP2 (G) or PP3 (Œ). Src family kinase inhibition by PP2 completely prevented ET-1 inhibition of Iamil, whereas ET-1 was fully inhibitory in the presence of PP3. Results are the average S.E. of five experiments. C, NIH 3T3 cells were treated with 10 nM ET-1 for 10 min and then lysed. Proteins were separated by SDS-PAGE and transferred to Immobilon-P. Total cellular tyrosine phosphorylation was assessed by Western blot using anti-phosphotyrosine antiserum (mono- clonal antibody clone 4G10). An increase in total cellular tyrosine phosphorylation was associated with ET-1 treatment. Results are representative of three separate experiments. D, NIH 3T3 cells were treated with 10 nM ET-1 for 10 min, then lysed and immunoprecipitated with 2 g of -Src family kinase antisera or rabbit IgG. Immunoprecipitates were subjected to in vitro kinase assays, and Src family kinase activity was assessed by phosphorylation of enolase. Lane 1 represents basal Src family kinase activity (no ET-1 treatment), which is enhanced upon stimulation with 10 nM ET-1 (lane 2). Pretreatment with PP2 blocked Src phosphorylation of enolase (lane 3). Lanes 4–6 are corresponding IgG controls. Results are representative of three separate experiments.

Article Snippet: In Vitro Kinase Assays—Kinases were immunoprecipitated from NIH 3T3 cell lysates using an antibody directed against the conserved COOH terminus of the Src family kinase sequence that recognizes all Src kinase family members (rabbit polyclonal antibody; Santa Cruz), or using antibodies to specific Src family kinases ( -Src kinase, Oncogene; -Yes kinase, Wako).

Techniques: Inhibition, In Vitro, Purification, Incubation, Modification, Transferring, Control, Phospho-proteomics, SDS Page, Western Blot, Immunoprecipitation, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Src Family Kinases Mediate Epithelial Na+ Channel Inhibition by Endothelin

doi: 10.1074/jbc.m106919200

Figure Lengend Snippet: FIG. 4. Activation of endogenous Src family kinases decreases ENaC currents. A, endogenous Src or Yes kinase was specifically immunoprecipitated from NIH 3T3 cell lysates, and their activities were assessed by in vitro phosphorylation of enolase. Immunoprecipitates incubated in the presence of phosphorylated, activating Src tail peptides (P) show increased phosphorylation of enolase, consistent with stimulation of Src and Yes kinase activity. Introduction of nonphosphorylated, control peptides (N) did not affect Src or Yes kinase activity. B, whole cell amiloride-sensitive currents were recorded from cells dialyzed with phosphorylated (Œ) or nonphosphorylated (G) Src tail peptides. In the presence of activating, phosphorylated peptides, Iamil was significantly decreased as compared with control (f no peptide). Results are average S.E. of four experiments.

Article Snippet: In Vitro Kinase Assays—Kinases were immunoprecipitated from NIH 3T3 cell lysates using an antibody directed against the conserved COOH terminus of the Src family kinase sequence that recognizes all Src kinase family members (rabbit polyclonal antibody; Santa Cruz), or using antibodies to specific Src family kinases ( -Src kinase, Oncogene; -Yes kinase, Wako).

Techniques: Activation Assay, Immunoprecipitation, In Vitro, Phospho-proteomics, Incubation, Activity Assay, Control