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: 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