Journal: Life Science Alliance

Article Title: Tyrosine phosphorylation of lamin A by Src promotes disassembly of nuclear lamina in interphase

doi: 10.26508/lsa.202101120

Figure Lengend Snippet: (A) MCF7 and MDA-MB-231 cells were treated with (+) or without (−) 50 nM dasatinib for 1 h and then lysed. Equal amounts of the whole cell lysates (WCLs) were incubated with anti-lamin A/C or pre-immune serum (IgG) as the control. The immunonocomplexes were analyzed by immunoblotting (IB) with anti-PY or anti-lamin A/C antibodies. An equal amount of WCLs was analyzed by immunoblotting with anti-Src, anti-Src pY416, or anti-actin. The tyrosine phosphorylation of lamin A was quantified and expressed as -fold relative to the level of MCF7 without dasatinib. (B) MCF7 and MDA-MB-231 cells were treated with (+) or without (−) 50 nM dasatinib for 1 h. The cells were fixed and stained for lamin A, lamin B1, and DNA. Representative images are shown. Scale bars, 10 μm. The nuclear circularity (4π × area/perimeter 2 ) was determined. The P -values were calculated from at least 150 cells pooled from three independent experiments. The percentage of the cells with nuclear lobulation was measured (n ≥ 400). The values (mean ± SD) are from three experiments. *** P < 0.001. (C) MDA-MB-231 cells were infected with lentivirues capable of expressing FLAG-lamin A or the mutants (Y45F and Y45D) and selected in the medium with neomycin (neo). An equal amount of WCLs was analyzed by immunoblotting (IB) with antibodies as indicated. (D) The cells as described in panel C were fixed and stained for FLAG-lamin A, lamin B1, and DNA. Representative images are shown. Scale bars, 10 μm. The percentage of the cells with nuclear lobulation was measured (n ≥ 900). Values (means ± SD) are from three experiments. * P < 0.05, ** P < 0.01, *** P < 0.001. Source data are available for this figure.

Article Snippet: The rabbit polyclonal anti-Src pY416 (MAB2685) antibody was purchased from R&D Systems.

Techniques: Incubation, Control, Western Blot, Phospho-proteomics, Staining, Infection, Expressing

Journal: Frontiers in Microbiology

Article Title: Candida albicans : The Ability to Invade Epithelial Cells and Survive under Oxidative Stress Is Unlinked to Hyphal Length

doi: 10.3389/fmicb.2017.01235

Figure Lengend Snippet: C. albicans SC5314 strain induced host cell biphasic ERK 1/2 activation and cortactin phosphorylation by SFKs. HeLa cells were incubated with blastospores from the SC5314 strain for the indicated time points. After incubation, HeLa cells were harvested, lysed, and their protein lysates separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Western blot using the indicated antibodies. The SC5314 strain induced (A) a biphasic activation of host ERK 1/2 (the other isolates did not, not shown) and also (B) the phosphorylation of cortactin (pCort [Y466]) by SFKs. These are representative observations from at least three independent experiments. SC5314, 997,5 g, oral L3837 and blood L3881 isolates induced host cell signaling activation. HeLa cells were incubated with blastospores from four C. albicans isolates for the indicated time points. After incubation, HeLa cells were harvested, lysed, and SFK and cortactin activation were analyzed by Western blot using the indicated antibodies. Cortactin, SFKs and actin have different molecular weights (80–85, 60, and 45 kDa, respectively), thus the nitrocellulose membrane was cut into three horizontal strips, blocked and each strip was incubated with anti-pY466 cortactin, anti-pY416 SFK or anti-actin. (C) pSFK (Y416) and (D) pCort (Y466), cortactin phosphorylated by Src at the Y 466 residue. Actin was used for protein loading normalization. Actin labeling is duplicated in (C,D) . pSFK/Act = densitometric rate of pSFK over actin. pCort/Act = densitometric rate of pCort over actin. These are representative observations from at least three independent experiments.

Article Snippet: Rabbit anti-pT202/Y204 ERK1/2 (#9101S) and rabbit anti-pY416 SFK (#2101S) were obtained from Cell Signaling, Beverly, MA, USA.

Techniques: Activation Assay, Phospho-proteomics, Incubation, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot, Membrane, Stripping Membranes, Residue, Labeling

Journal: Neuroscience

Article Title: Linkage of non-receptor tyrosine kinase Fyn to mGlu5 receptors in striatal neurons in a depression model

doi: 10.1016/j.neuroscience.2020.02.048

Figure Lengend Snippet: (A) Effects of social isolation on pY416, Fyn, and Src levels in the CPu. (B) Effects of social isolation on pY416, Fyn, and Src levels in the NAc. Note that social isolation (SI) had no significant effect on phosphorylation and expression of Fyn and Src in the CPu (A) and NAc (B) as compared to control (Con) rats. Representative immunoblots are shown left to the quantified data. Data were statistically analyzed using Student’s t-test (n = 6 per group) with ‘n’ equal to the number of animals.

Article Snippet: A rabbit antibody against pY416 (RRID:AB_10013641; Cell Signaling) reacts with the SFK members when phosphorylated at a conserved activation residue, a pan Y416 site.

Techniques: Isolation, Expressing, Western Blot

Journal: The Journal of Cell Biology

Article Title: Intramembrane binding of VE-cadherin to VEGFR2 and VEGFR3 assembles the endothelial mechanosensory complex

doi: 10.1083/jcb.201408103

Figure Lengend Snippet: VEcad TMD in flow signaling. (A) Requirement for VEcad. HUVECS were infected with scrambled or anti-VEcad shRNA-containing lentiviruses then subjected to 12 dynes/cm 2 laminar shear for 1 min. Activation of SFKs (SFK pY416 , ∼55 kD) and VEGFR2 (VEGFR2 pY1175 , ∼250 and 220 kD) were assayed by immunoblotting, with actin as a loading control. (B) VEcad TMD requirement for VEGFR activation. VEcad −/− cells reconstituted with VEcad WT , Ncad VE-TMD , Ncad WT , and VEcad N-TMD were subjected to laminar shear stress for 1 min, then VEGFR2 activation was assayed as in A. (C) VEcad requirement for PI3K signaling. Cells were subjected to laminar shear stress, then p85 immunoprecipitated and immunoblotted with anti-p85 pY458 antibody. Values beneath each panel indicate phosphorylation relative to cells without flow, quantified by densitometry with total p85 serving as a loading control. For all panels, values are means ± SEM, n = 3. IB, immunoblotting.

Article Snippet: Other phospho-antibodies used for immunoblotting include rabbit anti-SFK pY416 (#6943; Cell Signaling Technology), rabbit anti-p85 pY458 (#4228; Cell Signaling Technology), rabbit anti-Akt pS473 (700392; Invitrogen), and rabbit ant-p65 pS536 (#3033; Cell Signaling Technology).

Techniques: Infection, shRNA, Shear, Activation Assay, Western Blot, Control, Immunoprecipitation, Phospho-proteomics

Journal: REPRODUCTION

Article Title: Src family tyrosine kinase regulates acrosome reaction but not motility in porcine spermatozoa

doi: 10.1530/rep-11-0075

Figure Lengend Snippet: Figure 1 Identification of SRC family kinase (SFK) members in porcine spermatozoa. Semen samples from several boars were lysed and run in a 10% SDS–PAGE, and western blotting was performed using anti c-Lyn (A), anti c-Yes (B), and anti p-Y416 SFK (C) antibodies as described. Positive controls of different porcine tissues: liver, lung, and brain (A, lanes 4, 5 and 6), or somatic cell types such as rat pancreatic acini (B, lane 4) and neuroblasts (B, lane 5 and 6) were also included.

Article Snippet: FITC-PNA and anti-actin antibody were from Sigma–Aldrich; anti-Yes antibody was from BD Transduction Laboratories (Franklin Lakes, NJ, USA); anti-pY416 SFK was from Cell Signaling (Beverly, CA, USA); anti-Lyn antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); SU6656 and A23187 from Calbiochem (La Jolla, CA, USA); and FITC–phalloidin from Life Technologies.

Techniques: SDS Page, Western Blot

Journal: REPRODUCTION

Article Title: Src family tyrosine kinase regulates acrosome reaction but not motility in porcine spermatozoa

doi: 10.1530/rep-11-0075

Figure Lengend Snippet: Figure 2 Enzymatic activation of SFK during porcine spermatozoa capacitation. Spermatozoa samples incubated under noncapacitating (TBM) or capacitating conditions (TCM) during 4 h were lysated and run in a 10% SDS–PAGE, and western blotting analysis was performed using anti p-Y416 SFK or anti-actin as a protein loading control. Panel A shows a representative experiment for SFK1 and SFK2 tyrosine phosphorylation at the indicated times. Values shown at the bottom graphs in Panel B are meanGS.E.M. of three independent experiments. Quantification of bands was performed by scanning densitometry. Asterisk shows statistical differences between samples incubated under capacitating conditions in TCM with those incubated in TBM at the same time point.

Article Snippet: FITC-PNA and anti-actin antibody were from Sigma–Aldrich; anti-Yes antibody was from BD Transduction Laboratories (Franklin Lakes, NJ, USA); anti-pY416 SFK was from Cell Signaling (Beverly, CA, USA); anti-Lyn antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); SU6656 and A23187 from Calbiochem (La Jolla, CA, USA); and FITC–phalloidin from Life Technologies.

Techniques: Activation Assay, Incubation, SDS Page, Western Blot, Control, Phospho-proteomics

Journal: REPRODUCTION

Article Title: Src family tyrosine kinase regulates acrosome reaction but not motility in porcine spermatozoa

doi: 10.1530/rep-11-0075

Figure Lengend Snippet: Figure 3 SFK inhibition enhances spontaneous acrosome reaction in porcine spermatozoa. Spermatozoa samples were incubated under noncapacitating (TBM) or capacitating conditions (TCM) in the presence or absence of the SFK inhibitor SU6656 (10 mM) for 4 h. Flow cytometry was then performed using FITC-PNA as a specific marker for spermatozoa acrosome status and propidium iodide (PI) as a marker for cell death. Results are expressed as the percentage of PNA-positive and PI-negative spermatozoaGS.E.M. (nZ6). Asterisk shows statistical differences of samples incubated with or without SU6656 at the same time point.

Article Snippet: FITC-PNA and anti-actin antibody were from Sigma–Aldrich; anti-Yes antibody was from BD Transduction Laboratories (Franklin Lakes, NJ, USA); anti-pY416 SFK was from Cell Signaling (Beverly, CA, USA); anti-Lyn antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); SU6656 and A23187 from Calbiochem (La Jolla, CA, USA); and FITC–phalloidin from Life Technologies.

Techniques: Inhibition, Incubation, Flow Cytometry, Marker

Journal: REPRODUCTION

Article Title: Src family tyrosine kinase regulates acrosome reaction but not motility in porcine spermatozoa

doi: 10.1530/rep-11-0075

Figure Lengend Snippet: Figure 4 SFK inhibition enhances ionophore-induced acrosome reaction in porcine spermatozoa. Spermatozoa were incubated under capacitating conditions (TCM) with the ionophore A23187 (1 mM) in the presence or absence of the SFK inhibitor SU6656 (10 mM) for 4 h. Flow cytometry was then performed using FITC-PNA as a specific marker for spermatozoa acrosome status and propidium iodide (PI) as a marker for cell death. Results are expressed as the percentage of PNA-positive and PI-negative spermatozoaGS.E.M. (nZ4). Asterisk shows statistical differences of samples incubated with or without SU6656 at the same time point.

Article Snippet: FITC-PNA and anti-actin antibody were from Sigma–Aldrich; anti-Yes antibody was from BD Transduction Laboratories (Franklin Lakes, NJ, USA); anti-pY416 SFK was from Cell Signaling (Beverly, CA, USA); anti-Lyn antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); SU6656 and A23187 from Calbiochem (La Jolla, CA, USA); and FITC–phalloidin from Life Technologies.

Techniques: Inhibition, Incubation, Flow Cytometry, Marker

Journal: REPRODUCTION

Article Title: Src family tyrosine kinase regulates acrosome reaction but not motility in porcine spermatozoa

doi: 10.1530/rep-11-0075

Figure Lengend Snippet: Figure 5 Effect of SFK inhibition in the F-actin content of porcine spermatozoa under capacitating conditions. F-actin content of boar spermatozoa was evaluated by flow cytometry using FITC–phalloidin as a specific marker for F-actin, as described in the Materials and Methods section. (A) Shows typical cytograms obtained after FITC– phalloidin staining of spermatozoa under different conditions: noncapacitating (TCM time 0) and capacitating (TCM 2 h), and spermatozoa induced to undergo acrosome reaction with 10 mM calcium ionophore (TCM CA23187). Cytograms show an increase in FITC–phalloidin staining representative of F-actin formation after incubation on TCM for 2 h at 38.5 8C and that F-actin content decreases close to basal level after the induction of acrosome reaction. (B) Shows quantification by flow cytometry of F-actin content of spermatozoa incubated under capacitating conditions (TCM) in the absence (solid bars) or presence (striped bars) of the SFK inhibitor SU6656 (10 mM) for 4 h. Data are expressed as the average percentage of spermatozoa showing high fluorescence intensity (high FITC–phalloidin staining) GS.E.M. (nZ5). Asterisk shows statistical differences between sperma- tozoa incubated in the presence or absence of SU6656 at the same time.

Article Snippet: FITC-PNA and anti-actin antibody were from Sigma–Aldrich; anti-Yes antibody was from BD Transduction Laboratories (Franklin Lakes, NJ, USA); anti-pY416 SFK was from Cell Signaling (Beverly, CA, USA); anti-Lyn antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); SU6656 and A23187 from Calbiochem (La Jolla, CA, USA); and FITC–phalloidin from Life Technologies.

Techniques: Inhibition, Cytometry, Marker, Staining, Incubation

Journal: Environmental toxicology

Article Title: Gallic acid attenuates metastatic potential of human colorectal cancer cells through the miR-1247-3p-modulated integrin/FAK axis.

doi: 10.1002/tox.24087

Figure Lengend Snippet: FIGURE 5 Involvement of miR-1247-3p in the Gallic acid (GA)-inhibited integrin/FAK cascade in DLD-1 cells. Cells were treated with 90 μM GA for 24 h, lysed for total RNA extraction, and subjected to (A) miRNA expression profiling assay via microarray analysis or (B) quantitative analysis of the miRNAs via qPCR. miRNAs with up- and downregulation in response to GA were labeled with red and green, respectively. (C) Cells were transfected with control vector (Ctl-miR) or anti-miR-1247-3p (anti-miR), treated with GA for 24 h, and subjected to the assessment of integrins, FAK, and paxillin by Western blot. Densitometric analysis was performed for semi-quantitation of signals. The tubulin signal was used as internal control. Average signal ratios of phosphorylated protein/total protein compared with the control were indicated.

Article Snippet: Antibodies specifically recognizing human tubulin (SC-134237), integrin αV (SC-376156), integrin β3(SC-365679), focal adhesion kinase (FAK, SC-271126), phosphorylated FAK (pY397-FAK, SC-81493), paxillin (SC-136297), phosphorylated paxillin (pY118-Paxillin, SC-365020), Src (SC-32789), phosphorylated Src (pY416-Src, SC-24621, and CS#2101), and peroxidase-conjugated antibodies against mouse IgG or rabbit IgG were obtained from Santa Cruz Biotechnology, Inc. (CA, USA).

Techniques: RNA Extraction, Expressing, Microarray, Labeling, Transfection, Control, Plasmid Preparation, Western Blot, Quantitation Assay

Journal: Environmental toxicology

Article Title: Gallic acid attenuates metastatic potential of human colorectal cancer cells through the miR-1247-3p-modulated integrin/FAK axis.

doi: 10.1002/tox.24087

Figure Lengend Snippet: FIGURE 4 Gallic acid (GA) reduced integrin expression and inhibited FAK/Paxillin/Src and PI3K/AKT signaling in DLD-1 cells. Cells were treated with GA at the indicated concentrations for 24 h; collected; and lysed for the immunodetection of (A) integrins and the associated signaling proteins, (B) phosphor-paxillin and Src, and (C) PI3K, phosphor-AKT, and AKT via Western blot. Semi-quantitation of signals was conducted by densitometric analysis. DMSO treatment was used as control (C). Densitometric analysis was performed for the semi-quantitation of signals. The tubulin signal was used as internal control. Average signal ratios of phosphorylated protein/total protein compared with the control were indicated.

Article Snippet: Antibodies specifically recognizing human tubulin (SC-134237), integrin αV (SC-376156), integrin β3(SC-365679), focal adhesion kinase (FAK, SC-271126), phosphorylated FAK (pY397-FAK, SC-81493), paxillin (SC-136297), phosphorylated paxillin (pY118-Paxillin, SC-365020), Src (SC-32789), phosphorylated Src (pY416-Src, SC-24621, and CS#2101), and peroxidase-conjugated antibodies against mouse IgG or rabbit IgG were obtained from Santa Cruz Biotechnology, Inc. (CA, USA).

Techniques: Expressing, Immunodetection, Western Blot, Quantitation Assay, Control

Journal: American Journal of Physiology - Cell Physiology

Article Title: Serum starvation regulates E-cadherin upregulation via activation of c-Src in non-small-cell lung cancer A549 cells

doi: 10.1152/ajpcell.00132.2014

Figure Lengend Snippet: Serum starvation induces c-Src activity. A: c-Src activation was analyzed in A549 cells in serum-free media (0, 6 h, 24 h, 48 h) by immunoblotting for tyrosine 416 phosphorylation of c-Src (PY416-c-Src). Bottom panel: PY416-c-Src intensities were analyzed and normalized to β-actin intensities. *P < 0.05 compared with 0-h starvation data. B: A549 Phospho-c-Src Y416 analysis 24 h after exposure to a range of serum concentrations (0%, 0.5%, 1%, 5%, and 10 %) with PY416-c-Src intensities relative to β-actin intensities. *P < 0.05 compared with 0% FBS. C: immunocytochemistry of A549 cells cultured in media with or without serum for 24 h; IgG (green, top panel), PY416-c-Src (green, bottom panels), and DAPI (red) were visualized by fluorescence under equal exposure conditions. Representative immunoblots and images from 3 independent experiments are shown.

Article Snippet: Phospho-Src Y416 (pY416-Src) antibody was obtained from Cell Signaling Technology (Danvers, MA).

Techniques: Activity Assay, Activation Assay, Western Blot, Immunocytochemistry, Cell Culture, Fluorescence

Journal: American Journal of Physiology - Cell Physiology

Article Title: Serum starvation regulates E-cadherin upregulation via activation of c-Src in non-small-cell lung cancer A549 cells

doi: 10.1152/ajpcell.00132.2014

Figure Lengend Snippet: TGF-β1 attenuates serum starvation-mediated c-Src activity. A: A549 cells were starved for 0, 6 h, and 24 h in the presence of TGF-β1 (5 ng/ml) followed by PY416-c-Src immunoblotting. B: PY416-c-Src analysis of 24-h serum-starved A549 cells in the presence of TGF-β1 (0–5 ng/ml). C: PY416-c-Src (green) and DAPI (red) immunocytochemical analysis of A549 cells in serum-rich media (left), or serum-starved A549 cells in the absence (middle) or presence of TGF-β1 (5 ng/ml, right). Immunoblots and images shown are representative from 3 independent experiments.

Article Snippet: Phospho-Src Y416 (pY416-Src) antibody was obtained from Cell Signaling Technology (Danvers, MA).

Techniques: Activity Assay, Western Blot