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: Brain and Behavior

Article Title: Roles of adenosine A 1 receptors in the regulation of SFK activity in the rat forebrain

doi: 10.1002/brb3.2254

Figure Lengend Snippet: Primary Antibodies Used

Article Snippet: These include rabbit antibodies against Src (RRID:AB_2106047; Cell Signaling), Fyn (RRID:AB_631528; Santa Cruz Biotechnology, Santa Cruz, CA), phosphorylated Y416 (RRID:AB_10013641; pY416, Cell Signaling), or β‐actin (RRID:AB_476693; MilliporeSigma), or mouse antibodies against Src (RRID:AB_2106058; Cell Signaling), or Fyn (RRID:AB_627642; Santa Cruz).

Techniques: Concentration Assay, Blocking Assay, Recombinant, Phospho-proteomics

Journal: Brain and Behavior

Article Title: Roles of adenosine A 1 receptors in the regulation of SFK activity in the rat forebrain

doi: 10.1002/brb3.2254

Figure Lengend Snippet: Effects of 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX) on Src family kinase (SFK) Y416 phosphorylation in the rat striatum. (a) Effects of DPCPX on Y416 phosphorylation and expression of Fyn and Src in the caudate putamen (CPu). (b) Effects of DPCPX on Y416 phosphorylation and expression of Fyn and Src in the nucleus accumbens (NAc). Note that DPCPX at a higher rather than a lower dose significantly elevated pY416 levels in both the CPu and NAc, while DPCPX had no effect on total Fyn and Src expression. Representative immunoblots are shown to the left of the quantified data. Data are presented as means ± standard error of the mean ( n = 4 per group) with ‘n’ equal to the number of rats and were analyzed with one‐way ANOVA: CPu/pY416: F (2,9) = 6.400, n = 12, p = .019; CPu/Fyn: F (2,9) = 0.080, n = 12, p = .923; CPu/Src: F (2,9) = 0.317, n = 12, p = .736; NAc/pY416: F (2,9) = 7.771, n = 12, p = .011; NAc/Fyn: F (2,9) = 0.540, n = 12, p = .601; and NAc/Src: F (2,9) = 0.283, n = 12, p = .760. * p < .05 versus vehicle.

Article Snippet: These include rabbit antibodies against Src (RRID:AB_2106047; Cell Signaling), Fyn (RRID:AB_631528; Santa Cruz Biotechnology, Santa Cruz, CA), phosphorylated Y416 (RRID:AB_10013641; pY416, Cell Signaling), or β‐actin (RRID:AB_476693; MilliporeSigma), or mouse antibodies against Src (RRID:AB_2106058; Cell Signaling), or Fyn (RRID:AB_627642; Santa Cruz).

Techniques: Phospho-proteomics, Expressing, Western Blot

Journal: Brain and Behavior

Article Title: Roles of adenosine A 1 receptors in the regulation of SFK activity in the rat forebrain

doi: 10.1002/brb3.2254

Figure Lengend Snippet: Effects of 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX) on Src family kinase (SFK) Y416 phosphorylation in the rat medial prefrontal cortex (mPFC) and hippocampus. (a) Effects of DPCPX on Y416 phosphorylation and expression of Fyn and Src in the mPFC. (b) Effects of DPCPX on Y416 phosphorylation and expression of Fyn and Src in the hippocampus. Note that DPCPX at a higher rather than lower dose significantly elevated pY416 levels in the mPFC, while DPCPX had no effect on total Fyn and Src expression. No change in pY416 levels in the hippocampus was found following DPCPX administration. Representative immunoblots are shown to the left of the quantified data. Rats were given an i.p. injection of vehicle (0 mg/kg of DPCPX) or DPCPX (0.25 or 2.5 mg/kg) and were sacrificed 20 min after drug injection for immunoblot analysis. Data are presented as means ± standard error of the mean ( n = 4 per group) with ‘n’ equal to the number of rats and were analyzed with one‐way ANOVA: mPFC/pY416: F (2,9) = 4.884, n = 12, p = .036; mPFC/Fyn: F (2,9) = 0.443, n = 12, p = .655; mPFC/Src: F (2,9) = 0.003, n = 12, p = .998; hippocampus/pY416: F (2,9) = 0.290, n = 12, p = .755; hippocampus/Fyn: F (2,9) = 0.431, n = 12, p = .663; and hippocampus/Src: F (2,9) = 0.024, n = 12, p = .977. * p < .05 versus vehicle.

Article Snippet: These include rabbit antibodies against Src (RRID:AB_2106047; Cell Signaling), Fyn (RRID:AB_631528; Santa Cruz Biotechnology, Santa Cruz, CA), phosphorylated Y416 (RRID:AB_10013641; pY416, Cell Signaling), or β‐actin (RRID:AB_476693; MilliporeSigma), or mouse antibodies against Src (RRID:AB_2106058; Cell Signaling), or Fyn (RRID:AB_627642; Santa Cruz).

Techniques: Phospho-proteomics, Expressing, Western Blot, Injection

Journal: Brain and Behavior

Article Title: Roles of adenosine A 1 receptors in the regulation of SFK activity in the rat forebrain

doi: 10.1002/brb3.2254

Figure Lengend Snippet: Time‐dependent effects of 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX) on Src family kinase (SFK) Y416 phosphorylation in the rat caudate putamen (CPu). (a) Representative immunoblots illustrating time‐dependent effects of DPCPX on Y416 phosphorylation and expression of Fyn and Src in the CPu. (b–d) Quantifications of effects of DPCPX on Y416 phosphorylation (b) and expression of Fyn (c) and Src (d) in the CPu. Note that DPCPX induced a time‐dependent increase in pY416 levels, while DPCPX had no effect on total Fyn and Src expression. Rats were given an i.p. injection of vehicle or DPCPX (2.5 mg/kg) and were sacrificed at different time points (20 min, 1 h, and 3 h) after drug injection for immunoblot analysis. Data are presented as means ± standard error of the mean ( n = 4–5 per group) with ‘n’ equal to the number of animals and were analyzed with unpaired Student's t ‐test: pY416/20 min: t (8) = 2.428, n = 10, p = .041; pY416/1 h: t (6) = 6.286, n = 8, p < .001; pY416/3 h: t (6) = 0.357, n = 8, p = .998; Fyn/20 min: t (8) = 0.590, n = 10, p = .561; Fyn/1 h: t (6) = 1.613, n = 8, p = .158; Fyn/3 h: t (6) = 0.818, n = 8, p = .445; Src/20 min: t (8) = 0.495, n = 10, p = .634; Src/1 h: t (6) = .556, n = 8, p = .598; and Src/3 h: t (6) = 0.162, n = 8, p = .876. * p < .05 versus vehicle at the same time points.

Article Snippet: These include rabbit antibodies against Src (RRID:AB_2106047; Cell Signaling), Fyn (RRID:AB_631528; Santa Cruz Biotechnology, Santa Cruz, CA), phosphorylated Y416 (RRID:AB_10013641; pY416, Cell Signaling), or β‐actin (RRID:AB_476693; MilliporeSigma), or mouse antibodies against Src (RRID:AB_2106058; Cell Signaling), or Fyn (RRID:AB_627642; Santa Cruz).

Techniques: Phospho-proteomics, Western Blot, Expressing, Injection

Journal: Brain and Behavior

Article Title: Roles of adenosine A 1 receptors in the regulation of SFK activity in the rat forebrain

doi: 10.1002/brb3.2254

Figure Lengend Snippet: Time‐dependent effects of 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX) on Src family kinase (SFK) Y416 phosphorylation in the rat nucleus accumbens (NAc). (a) Representative immunoblots illustrating time‐dependent effects of DPCPX on Y416 phosphorylation and expression of Fyn and Src in the NAc. (b–d) Quantifications of effects of DPCPX on Y416 phosphorylation (b) and expression of Fyn (c) and Src (d) in the NAc. Note that DPCPX induced a time‐dependent increase in pY416 levels, while DPCPX had no effect on total Fyn and Src expression. Rats were given an i.p. injection of vehicle or DPCPX (2.5 mg/kg) and were sacrificed at different time points (20 min, 1 h, and 3 h) after drug injection for immunoblot analysis. Data are presented as means ± standard error of the mean ( n = 4–5 per group) with ‘n’ equal to the number of animals and were analyzed with unpaired Student's t ‐test: pY416/20 min: t (8) = 3.292, n = 10, p = .011; pY416/1 h: t (6) = 3.828, n = 8, p = .009; pY416/3 h: t (6) = 0.042, n = 8, p = .968; Fyn/20 min: t (8) = 0.143, n = 10, p = .890; Fyn/1 h: t (6) = 0.530, n = 8, p = .615; Fyn/3 h: t (6) = 0.649, n = 8, p = .540; Src/20 min: t (8) = 0.660, n = 10, p = .528; Src/1 h: t (6) = 0.123, n = 8, p = .906; and Src/3 h: t (6) = 0.793, n = 8, p = .458. * p < .05 versus vehicle at the same time points.

Article Snippet: These include rabbit antibodies against Src (RRID:AB_2106047; Cell Signaling), Fyn (RRID:AB_631528; Santa Cruz Biotechnology, Santa Cruz, CA), phosphorylated Y416 (RRID:AB_10013641; pY416, Cell Signaling), or β‐actin (RRID:AB_476693; MilliporeSigma), or mouse antibodies against Src (RRID:AB_2106058; Cell Signaling), or Fyn (RRID:AB_627642; Santa Cruz).

Techniques: Phospho-proteomics, Western Blot, Expressing, Injection

Journal: Brain and Behavior

Article Title: Roles of adenosine A 1 receptors in the regulation of SFK activity in the rat forebrain

doi: 10.1002/brb3.2254

Figure Lengend Snippet: Time‐dependent effects of 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX) on Src family kinase (SFK) Y416 phosphorylation in the rat medial prefrontal cortex (mPFC). (a) Representative immunoblots illustrating time‐dependent effects of DPCPX on Y416 phosphorylation and expression of Fyn and Src in the mPFC. (b–d) Quantifications of effects of DPCPX on Y416 phosphorylation (b) and expression of Fyn (c) and Src (d) in the mPFC. Note that DPCPX induced a time‐dependent increase in pY416 levels, while DPCPX had no effect on total Fyn and Src expression. Rats were given an i.p. injection of vehicle or DPCPX (2.5 mg/kg) and were sacrificed at different time points (20 min, 1 h, and 3 h) after drug injection for immunoblot analysis. Data are presented as means ± standard error of the mean ( n = 4–5 per group) with ‘n’ equal to the number of animals and were analyzed with unpaired Student's t ‐test: pY416/20 min: t (8) = 3.593, n = 10, p = .01; pY416/1 h: t (6) = 2.732, n = 8, p = .034; pY416/3 h: t (6) = 0.335, n = 8, p = .749; Fyn/20 min: t (8) = 0.329, n = 10, p = .751; Fyn/1 h: t (6) = 0.278, n = 8, p = .790; Fyn/3 h: t (6) = 0.707, n = 8, p = .506; Src/20 min: t (8) = 0.418, n = 10, p = .687; Src/1 h: t (6) = .179, n = 8, p = .864; and Src/3 h: t (6) = 0.918, n = 8, p = .394. * p < .05 versus vehicle at the same time points.

Article Snippet: These include rabbit antibodies against Src (RRID:AB_2106047; Cell Signaling), Fyn (RRID:AB_631528; Santa Cruz Biotechnology, Santa Cruz, CA), phosphorylated Y416 (RRID:AB_10013641; pY416, Cell Signaling), or β‐actin (RRID:AB_476693; MilliporeSigma), or mouse antibodies against Src (RRID:AB_2106058; Cell Signaling), or Fyn (RRID:AB_627642; Santa Cruz).

Techniques: Phospho-proteomics, Western Blot, Expressing, Injection

Journal: Brain and Behavior

Article Title: Roles of adenosine A 1 receptors in the regulation of SFK activity in the rat forebrain

doi: 10.1002/brb3.2254

Figure Lengend Snippet: Effects of 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX) on Y416 phosphorylation of immunopurified Src family kinase (SFK) members in the rat striatum and medial prefrontal cortex (mPFC). (a and b) Effects of DPCPX on Y416 phosphorylation of immunopurified Fyn (a) and Src (b) proteins in the striatum. (c and d) Effects of DPCPX on Y416 phosphorylation of immunopurified Fyn (c) and Src (d) proteins in the mPFC. Note that DPCPX enhanced pY416 levels in Fyn but not Src in the striatum and in both Fyn and Src in the mPFC. Representative immunoblots (IB) are shown to the left of the quantified data. Rats were given an i.p. injection of vehicle or DPCPX (2.5 mg/kg) and were sacrificed 20–30 min after drug injection. Fyn and Src proteins were purified from the striatum and mPFC via immunoprecipitation (IP) with a specific antibody indicated. Data are presented as means ± standard error of the mean ( n = 4 per group) with ‘n’ equal to the number of animals and were analyzed with unpaired Student's t ‐test: striatum (pY416/Fyn): t (6) = 3.679, n = 8, p = .010; striatum (pY416/Src): t (6) = 1.626, n = 8, p = .155; mPFC (pY416/Fyn): t (6) = 3.016, n = 8, p = .023; and mPFC (pY416/Src): t (6) = 3.553, n = 8, p = .012. * p < .05 versus vehicle.

Article Snippet: These include rabbit antibodies against Src (RRID:AB_2106047; Cell Signaling), Fyn (RRID:AB_631528; Santa Cruz Biotechnology, Santa Cruz, CA), phosphorylated Y416 (RRID:AB_10013641; pY416, Cell Signaling), or β‐actin (RRID:AB_476693; MilliporeSigma), or mouse antibodies against Src (RRID:AB_2106058; Cell Signaling), or Fyn (RRID:AB_627642; Santa Cruz).

Techniques: Phospho-proteomics, Western Blot, Injection, Purification, Immunoprecipitation

Journal: The Journal of Neuroscience

Article Title: Protein Tyrosine Phosphatase δ Mediates the Sema3A-Induced Cortical Basal Dendritic Arborization through the Activation of Fyn Tyrosine Kinase

doi: 10.1523/JNEUROSCI.2519-16.2017

Figure Lengend Snippet: Antibodies

Article Snippet: pY416 Src (D49G4), rabbit mAb , Cell Signaling Technology (9935, 6943) RRID: AB_10860245 , 1:5000 , IB.

Techniques: Labeling

Journal: The Journal of Neuroscience

Article Title: Protein Tyrosine Phosphatase δ Mediates the Sema3A-Induced Cortical Basal Dendritic Arborization through the Activation of Fyn Tyrosine Kinase

doi: 10.1523/JNEUROSCI.2519-16.2017

Figure Lengend Snippet: Dephosphorylation C-terminal Tyr527 residues of Src and Fyn by PTPδ. A, Hyperphosphorylated proteins in Ptpδ mutant mice. Whole-brain lysates from Ptpδ mutant mice were blotted with anti-phosphotyrosine mAb (4G10). Two immunoreactive bands, 100 and 60 kDa, are hyperphosphorylated in Ptpδ−/− compared with Ptpδ+/−. B, Phosphorylation of Src and Fyn. Immunoprecipitated Src and Fyn from the indicated mutant mice were blotted with anti-phosphotyrosine mAb. Both Src and Fyn are hyperphosphorylated in Ptpδ−/−. C, Hyperphosphorylation of Y527-Src in Ptpδ−/−. Lysates from the indicated mutant mice were blotted with anti-phospho-Y416-Src (pY416) or anti-phospho-Y527-Src (pY527)-specific antibodies. The phosphorylation of Y527 is increased in Ptpδ−/− brains. D, Sema3A-induced dephosphorylation of pY527-Src in DRG growth cones. The neurons from E16–E18 Ptpδ mutant mice were stimulated with AP-Sema3A (0.1 nm) and were double-immunostained with anti-pY527-specific pAb (green) and anti-Src mAb (red). Sema3A-stimulation decreases pY527 in Ptpδ+/− neurons, but not in Ptpδ−/−. Scale bar, 10 μm. E, Scored graph. The intensity of immunoreactive signals within the growth cones was analyzed. The ratio of pY527 and Src (pY527/Src) in nonstimulated growth cones was treated as 100%. Each point represents the average ± SEM (n = 5, independent experiments). The pY527/Src ratio in wt and Ptpδ+/− (black solid line) is decreased after Sema3A-stimulation, whereas the ratio in Ptpδ−/− (red dashed line) is relatively unchanged. Unpaired t test between +/+, +/−, and −/− (5 min: t(8) = −3.57, p = 0.0073; 10 min: t(8) = −4.02, p = 0.0038; 30 min: t(8) = −3.16, p = 0.013). *p < 0.05, **p < 0.01. F, G, DRG neurons from Ptpδ+/−; Fyn+/− double-heterozygous are less sensitive to Sema3A stimulation. One-way ANOVA with post hoc Tukey–Kramer test (wt: 7; Ptpδ+/−: 7; Fyn+/−: 7; Ptpδ+/−; Fyn+/−: 13 explants from 3 independent experiments). *p < 0.05. Scale bar, 10 μm.

Article Snippet: pY416 Src (D49G4), rabbit mAb , Cell Signaling Technology (9935, 6943) RRID: AB_10860245 , 1:5000 , IB.

Techniques: De-Phosphorylation Assay, Mutagenesis, Phospho-proteomics, Immunoprecipitation

Journal: Immunology

Article Title: Increased sensitivity to antigen in high avidity CD8 + T cells results from augmented membrane proximal T-cell receptor signal transduction

doi: 10.1111/j.1365-2567.2011.03440.x

Figure Lengend Snippet: The amount of peptide required for p56Lck phosphorylation reflects that required for effector function. (a) The level of Src-family kinase p56Lck was similar in high and low avidity cytotoxic T lymphocytes (CTL). Cells were intracellularly stained with p56Lck specific antibodies. Thin and bold lines represent low and high avidity CTL respectively, whereas shaded histograms represent isotype control staining. (b) CTL lines were stimulated for 10 min with antigen-presenting cells pulsed with different concentrations of peptides. Cell lysates were run on 10% SDS–PAGE and blotted to nitrocellulose membrane. Membranes were exposed to anti-phospho Lck antibody (pY416) followed by horseradish peroxidase-conjugated anti-rabbit secondary antibodies. Blots were developed and quantified by densitometry. Data are representative of three independent experiments.

Article Snippet: For measurement of phosphorylation of p56 Lck , stimulated cells were lysed and transferred to the nitrocellulose membrane before probing them with specific antibodies (PY 416; Cell Signaling) and blots were developed as described above.

Techniques: Phospho-proteomics, Staining, Control, SDS Page, Membrane