Journal: PLoS ONE

Article Title: HIF-1 and c-Src Mediate Increased Glucose Uptake Induced by Endothelin-1 and Connexin43 in Astrocytes

doi: 10.1371/journal.pone.0032448

Figure Lengend Snippet: A ) Astrocytes were incubated in the absence (control, C) or presence of 0.1 µM ET-1 for the indicated times, proteins were extracted and Y416 c-Src and total c-Src were analysed by Western blot. The results are expressed as percentages of the level found in the control and they show that ET-1 rapidly and transiently increased Y416 c-Src without affecting significantly total c-Src. ***p<0.001; *p<0.05 versus control. B ) Astrocytes were transfected with NT-siRNA or with Cx43-siRNA. After 48 h, proteins were extracted and Y416 c-Src, total c-Src and Cx43 were analysed by Western blot. The results are expressed as percentages of the level found in the control and they show that silencing Cx43 increased Y416 c-Src without affecting significantly total c-Src. **p<0.01 versus NT-siRNA.

Article Snippet: Rabbit polyclonal antibody against total c-Src (2108) and rabbit polyclonal antibody against Y416 c-Src (2101) were from Cell Signaling Technology (Boston, EEUU).

Techniques: Incubation, Western Blot, Transfection

Journal: PLoS ONE

Article Title: HIF-1 and c-Src Mediate Increased Glucose Uptake Induced by Endothelin-1 and Connexin43 in Astrocytes

doi: 10.1371/journal.pone.0032448

Figure Lengend Snippet: Astrocytes were preincubated with 100 ng/µL PP2 (c-Src inhibitor) or 100 ng/µL PP3 (inactive analogue) for 1 h and these agents were maintained for the rest of the experiment. Then, cells were incubated in the absence or presence of 0.1 µM ET-1 for the indicated times and proteins were extracted for the analysis of Y416 c-Src and total c-Src by Western blot. The results are expressed as percentages of the level found in the control (PP3, time 0) and they show that PP2 inhibited the activation of c-Src (Y416 c-Src) promoted by ET-1. ***p<0.001; *p<0.05 versus the corresponding time 0.

Article Snippet: Rabbit polyclonal antibody against total c-Src (2108) and rabbit polyclonal antibody against Y416 c-Src (2101) were from Cell Signaling Technology (Boston, EEUU).

Techniques: Incubation, Western Blot, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Connexin43 Region 266–283, via Src Inhibition, Reduces Neural Progenitor Cell Proliferation Promoted by EGF and FGF-2 and Increases Astrocytic Differentiation

doi: 10.3390/ijms21228852

Figure Lengend Snippet: Effect of TAT-Cx43 266–283 on neural progenitor cell proliferation, apoptosis, and Src activity in proliferation conditions. ( A , B ) Representative epifluorescence images and quantifications of Ki67 ( A , in red) or caspase-3 ( B , in red) immunostaining in cultures of neural progenitor cells in proliferation conditions (PC NPCs) treated with 25 μM TAT or TAT-Cx43 266–283 for 72 h. Cell nuclei of the same fields were visualized by DAPI staining (turquoise). Bar: 75 µm. Results are expressed as percentage of Ki67-positive cells (in A ) or caspase-3-positive cells (in B ) and are the mean ± SEM ( n = 3; * p < 0.05; ** p < 0.01; Student’s t -test). ( C ) Western blotting showing Y416 Src and total Src in PC NPCs treated with 25 µM TAT or TAT-Cx43 266–283 for 72 h. Quantification of Y416 Src/Src ratio expressed as a percentage with respect to TAT. Results are expressed as mean ± SEM ( n = 4; *** p < 0.001; Student’s t -test).

Article Snippet: After blocking, the membranes were incubated overnight at 4 °C with the primary antibodies against rabbit Y416 Src (Cell Signaling; Ref. 2101; 1:200), rabbit total c-Src (Cell Signaling; Ref. 2108; 1:500), guinea pig DCX (Chemicon International, Merck Millipore, Madrid Spain; Ref. AB5910; 1:1000), mouse GFAP (Sigma; Ref. G3893; 1:500), or mouse β catenin (BD Bioscience, Madrid, Spain; Ref. 610153; 1:500).

Techniques: Activity Assay, Immunostaining, Staining, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Connexin43 Region 266–283, via Src Inhibition, Reduces Neural Progenitor Cell Proliferation Promoted by EGF and FGF-2 and Increases Astrocytic Differentiation

doi: 10.3390/ijms21228852

Figure Lengend Snippet: Involvement of Src and β-catenin in the effects of TAT-Cx43 266–283 on neural progenitor cell differentiation. ( A ) Western blots showing Y416 Src and total Src in neural progenitor cells treated with 25 µM of TAT or TAT-Cx43 266–283 in differentiation conditions for 96 h. Quantification of Y416 Src/Src ratio expressed as a percentage of TAT. Results are the mean ± SEM ( n = 5; ** p < 0.01; Student’s t -test). ( B ) Representative images showing doublecortin (DCX) immunoreactivity (red) in neural progenitor cells in differentiation conditions (DC NPCs) after 96 h of treatment with DMSO or with 1 µM Dasatinib. Cell nuclei were labeled with DAPI (blue). Bar: 50 µm. Percentage of DCX-positive cells in both experimental conditions. Results are shown as mean ± SEM ( n = 5; ** p < 0.01; Student’s t -test). ( C ) Representative images showing glial fibrillary acidic protein (GFAP) immunoreactivity (green) in DC NPCs after 96 h of treatment with DMSO or with 1 µM Dasatinib. Cell nuclei are labeled with DAPI (blue). Bar: 50 µm. Percentage of GFAP-positive cells in both experimental conditions. Results are shown as mean ± SEM ( n = 6; non-significant differences; Student’s t -test). ( D ) β-catenin levels were analyzed by western blotting in DC NPCs treated with 25 µM of TAT or TAT-Cx43 266–283 for 96 h. The bar graph shows the ratio of β-catenin/GAPDH in percentage with respect to TAT (mean ± SEM; n = 3; ** p < 0.01; Student’s t -test).

Article Snippet: After blocking, the membranes were incubated overnight at 4 °C with the primary antibodies against rabbit Y416 Src (Cell Signaling; Ref. 2101; 1:200), rabbit total c-Src (Cell Signaling; Ref. 2108; 1:500), guinea pig DCX (Chemicon International, Merck Millipore, Madrid Spain; Ref. AB5910; 1:1000), mouse GFAP (Sigma; Ref. G3893; 1:500), or mouse β catenin (BD Bioscience, Madrid, Spain; Ref. 610153; 1:500).

Techniques: Cell Differentiation, Western Blot, Labeling

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Pyk2 phosphorylation of VE-PTP downstream of STIM1-induced Ca 2+ entry regulates disassembly of adherens junctions

doi: 10.1152/ajplung.00008.2017

Figure Lengend Snippet: COOH-terminal tyrosine phosphorylation of VE-PTP triggers Src activation in ECs. A: confluent HLMVECs pretreated with Src inhibitor 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) (10 μM) for 30 min were exposed to thrombin for different time intervals. Then cells were used to determine VE-cadherin phosphorylation at Y685 or Y731. Quantified results shown are from 3 experiments as the ratio of phosphorylated:total protein. **P < 0.01; ***P < 0.001; control vs. PP1-treated. B: HLMVECs transfected with sc-siRNA or Pyk2-siRNA were treated with thrombin and immunoblotted with an antibody specific to phospho-Y416-Src. Quantified results shown are from 3 experiments as the ratio of phosphorylated:total protein. **P < 0.01; sc-siRNA vs. Pyk2-siRNA. C: C57BL/6 mice injected with sc-siRNA or Pyk2-siRNA (see above in Fig. 4) were intravenously injected with PAR-1 agonist peptide for 0, 20, and 60 min. Lung extracts prepared after PAR-1 agonist peptide treatment were immunoprecipitated with anti-VE-PTP pAb. The immunoprecipitated samples were immunoblotted with anti-Src mAb. Quantified results shown are from 3 experiments. **P < 0.01; sc-siRNA vs. Pyk2-siRNA. D: HLMVECs treated with Pyk2 inhibitor followed by thrombin exposure were immunoprecipitated with anti-VE-PTP pAb. The immunoprecipitated samples were immunoblotted with anti-Src mAb. Quantified results shown are from 3 separate experiments. *P < 0.05; vehicle vs. PF431396. E: confluent HLMVECs were incubated for 60 min with the indicated concentrations of either cell-permeable WT-VE-PTP or Mut-VE-PTP peptide and then exposed to thrombin for 30 min. Cell lysates were then immunoblotted with an antibody specific to phos-Y416-Src. Results are means ± SE of 3 experiments quantified as the ratio of phosphorylated:total protein. *P < 0.05; **P < 0.01; control (vehicle-treated) vs. WT-VE-PTP peptide-treated. F: HLMVECs, pretreated with WT-VE-PTP or Mut-VE-PTP peptide and then exposed to thrombin, were immunoprecipitated with anti-VE-PTP pAb. The immunoprecipitated samples were immunoblotted with anti-phosphotyrosine mAb. Results are means ± SE of 3 experiments quantified as the ratio of phosphorylated to total protein. *P < 0.05; **P < 0.01; control (vehicle-treated) vs. WT-VE-PTP peptide-treated.

Article Snippet: Rabbit pAb specific to phospho-Pyk2 Y402 (cat. no. 3291) ( 48 ), rabbit pAb against Pyk2 (cat. no. 3090) ( 32 ), rabbit mAb against phospho- Src Y416 (cat. no. 6943) ( 24 ), and rabbit mAb against Src (cat. no. 2123) ( 3 ) were purchased from Cell Signaling (Danvers, MA).

Techniques: Activation Assay, Transfection, Injection, Immunoprecipitation, Incubation

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Pyk2 phosphorylation of VE-PTP downstream of STIM1-induced Ca 2+ entry regulates disassembly of adherens junctions

doi: 10.1152/ajplung.00008.2017

Figure Lengend Snippet: Model for induction of VE-PTP-dependent Src activation in ECs via PAR-1-SOCE-Pyk2 pathway. PAR-1-induced ER store Ca2+ depletion via phospholipase C (PLC)-inositol 1,4,5-triphosphate (IP3) activates SOCE, inducing autophosphorylation of Pyk2 at Y402 to activate Pyk2. The activated Pyk2 binds to and phosphorylates VE-PTP at Y1981 in the COOH-terminal region. The phospho-Y1981 of VE-PTP binds to the Src homology-2 (SH2) domain of Src (anchored to the membrane), which, in turn, promotes the autophosphorylation of Y416 in the kinase domain of Src to trigger Src activation. The activated Src can phosphorylate tyrosine residues in the cytosolic COOH-terminal region of VE-cadherin to promote endothelial barrier dysfunction.

Article Snippet: Rabbit pAb specific to phospho-Pyk2 Y402 (cat. no. 3291) ( 48 ), rabbit pAb against Pyk2 (cat. no. 3090) ( 32 ), rabbit mAb against phospho- Src Y416 (cat. no. 6943) ( 24 ), and rabbit mAb against Src (cat. no. 2123) ( 3 ) were purchased from Cell Signaling (Danvers, MA).

Techniques: Activation Assay