phosphorylated src family kinase y416  (Cell Signaling Technology Inc)

Journal: British journal of pharmacology

Article Title: Exploring the pathophysiology underlying clozapine-induced enhancement of glutamatergic transmission through L-glutamate and D-serine release associated with pannexin1 hemichannels.

doi: 10.1111/bph.70112

Figure Lengend Snippet: FIGURE 2 Dose-dependent effects of chronic administration of clozapine on expression of pannexin1 and phosphorylated Src in the mPFC. Rats were chronically administered with clozapine (0, 5 and 10 mg kg1 day1 for 28 days). In the upper histograms, ordinate: mean ± SD (n = 6) of the relative levels of pannexin1 (PANX) per GAPDH (a) and phosphorylated-Src (pSrc) per Src (b). *P < 0.05: relative to control (clozapine free) by one-way ANOVA with Scheffe's post hoc test. F-values of the chronic clozapine administration on the expression of pannexin1 and pSrc in the rat frontal cortex using one-way ANOVA were [F(3,15) = 17.3 (P < 0.01)] and [F(3,15) = 18.0 (P < 0.01)], respectively. The lower panels indicate pseudo-gel images of capillary immunoblotting.

Article Snippet: Primary antibodies against GAPDH (NB300-327, RRID:AB_10001915, 1:300; Novus Biologicals, Littleton, CO, USA), pannexin1 (12595-1-AP, RRID:AB_ 2159186, 1:100; Proteintech, Rosemont, IL, USA), Src (2109, RRID: AB_2106059,1:50; Cell Signalling Technology, Danvers, MA, USA) and phosphorylated Src (pSrc; MAB2685, RRID:AB_3657975, 1 μg ml 1, R&D Systems, Minneapolis, MN, USA) were diluted in an antibody diluent (Immuno Shot Platinum; CosmoBio, Tokyo, Japan).

Techniques: Expressing, Control, Western Blot

Journal: British journal of pharmacology

Article Title: Exploring the pathophysiology underlying clozapine-induced enhancement of glutamatergic transmission through L-glutamate and D-serine release associated with pannexin1 hemichannels.

doi: 10.1111/bph.70112

Figure Lengend Snippet: FIGURE 6 Concentration-dependent effects of chronic administration of clozapine on expression of pannexin1 and phosphorylated Src in cultured astrocytes. (a and b) Concentration-dependent effects of chronic exposure to clozapine (3 and 10 μM for 28 days) on expression of pannexin1 and phosphorylated Src in the astroglial plasma membrane fraction, respectively. In the upper histograms, ordinate: mean ± SD (n = 6) of the relative levels of pannexin1 (PANX) per GAPDH or phosphorylated Src (pSrc) per Src. *P < 0.05: relative to control (clozapine free) by one- way ANOVA with Scheffe's post hoc test. F-values of the concentration-dependent effects of chronic clozapine administration on the expression of pannexin1 and pSrc using one-way ANOVA were [F(2,15) = 21.3 (P < 0.01)] and [F(2,15) = 14.9 (P < 0.01)], respectively. The lower panels indicate pseudo-gel images of capillary immunoblotting.

Article Snippet: Primary antibodies against GAPDH (NB300-327, RRID:AB_10001915, 1:300; Novus Biologicals, Littleton, CO, USA), pannexin1 (12595-1-AP, RRID:AB_ 2159186, 1:100; Proteintech, Rosemont, IL, USA), Src (2109, RRID: AB_2106059,1:50; Cell Signalling Technology, Danvers, MA, USA) and phosphorylated Src (pSrc; MAB2685, RRID:AB_3657975, 1 μg ml 1, R&D Systems, Minneapolis, MN, USA) were diluted in an antibody diluent (Immuno Shot Platinum; CosmoBio, Tokyo, Japan).

Techniques: Concentration Assay, Expressing, Cell Culture, Clinical Proteomics, Membrane, Control, Western Blot

Journal: British journal of pharmacology

Article Title: Exploring the pathophysiology underlying clozapine-induced enhancement of glutamatergic transmission through L-glutamate and D-serine release associated with pannexin1 hemichannels.

doi: 10.1111/bph.70112

Figure Lengend Snippet: FIGURE 7 Interaction among clozapine and inhibitors of Src and III-mGluR on the expression of pannexin1 and phosphorylated Src in the cultured astrocytes. (a) Interaction between 10 μM PP2 (Src inhibitor) and 10 μM clozapine on pannexin1 expression in the astroglial plasma membrane. (b) Interaction between 100 μM CPPG (III-mGluR inhibitor) and 10 μM clozapine on phosphorylated-Src expression in the astroglial plasma membrane. In the upper histograms, ordinate: mean ± SD (n = 6) of the relative levels of pannexin1 (PANX) per GAPDH or phosphorylated Src (pSrc) per Src. *P < 0.05: relative to control (clozapine free) by two-way ANOVA with Scheffe's post hoc test. @: P < 0.05: relative to 10 μM clozapine exposure by two-way ANOVA with Scheffe's post hoc test. F-values of the interaction between 10 μM PP2 and chronic exposure to 10 μM clozapine on the expression of pannexin1 using two-way ANOVA were [Fclozapine(1,20) = 55.5 (P < 0.01), FPP2(1,20) = 0.2 (P > 0.05), Fclozapine*PP2(1,20) = 1.1 (P > 0.05)]. F- values of the interaction between 100 μM CPPG and chronic exposure to 10 μM clozapine on the expression of pSrc using two-way ANOVA were [Fclozapine(1,20) = 56.0 (P < 0.01), FCPPG(1,20) = 27.7 (P < 0.01), Fclozapine*CPPG(1,20) = 0.4 (P > 0.05)]. The lower panels indicate pseudo-gel images of capillary immunoblotting.

Article Snippet: Primary antibodies against GAPDH (NB300-327, RRID:AB_10001915, 1:300; Novus Biologicals, Littleton, CO, USA), pannexin1 (12595-1-AP, RRID:AB_ 2159186, 1:100; Proteintech, Rosemont, IL, USA), Src (2109, RRID: AB_2106059,1:50; Cell Signalling Technology, Danvers, MA, USA) and phosphorylated Src (pSrc; MAB2685, RRID:AB_3657975, 1 μg ml 1, R&D Systems, Minneapolis, MN, USA) were diluted in an antibody diluent (Immuno Shot Platinum; CosmoBio, Tokyo, Japan).

Techniques: Expressing, Cell Culture, Clinical Proteomics, Membrane, Control, Western Blot

Journal: British journal of pharmacology

Article Title: Exploring the pathophysiology underlying clozapine-induced enhancement of glutamatergic transmission through L-glutamate and D-serine release associated with pannexin1 hemichannels.

doi: 10.1111/bph.70112

Figure Lengend Snippet: FIGURE 9 Effects of chronic administration of L-BAIBA on Src signalling in the cultured astrocytes. (a and b) Interaction among chronic exposure to 30 μM L-BAIBA for 28 days and inhibitors of III-mGluR (100 μM CPPG) and NMDAR (1 μM MK801), respectively. In the upper side histogram, ordinate: mean ± SD (n = 6) of the relative levels of phosphorylated-Src (pSrc) per Src. *P < 0.05: relative to control (L-BAIBA free) by two-way ANOVA with Scheffe's post hoc test. @: P < 0.05: relative to 30 μM L-BAIBA exposure alone. F-values of the interaction between 100 μM CPPG and chronic exposure to 30 μM L-BAIBA on the expression of pSrc using two-way ANOVA were [FL-BAIBA(1,20) = 27.6 (P < 0.01), FCPPG(1,20) = 59.3 (P < 0.01), FL-BAIBA*CPPG(1,20) = 2.7 (P > 0.05)]. F-values of the interaction between 1 μM MK801 and chronic exposure to 30 μM L-BAIBA on the expression of pSrc using two-way ANOVA were [FL-BAIBA(1,20) = 46.3 (P < 0.01), FMK801(1,20) = 1.0 (P > 0.05), FL-

Article Snippet: Primary antibodies against GAPDH (NB300-327, RRID:AB_10001915, 1:300; Novus Biologicals, Littleton, CO, USA), pannexin1 (12595-1-AP, RRID:AB_ 2159186, 1:100; Proteintech, Rosemont, IL, USA), Src (2109, RRID: AB_2106059,1:50; Cell Signalling Technology, Danvers, MA, USA) and phosphorylated Src (pSrc; MAB2685, RRID:AB_3657975, 1 μg ml 1, R&D Systems, Minneapolis, MN, USA) were diluted in an antibody diluent (Immuno Shot Platinum; CosmoBio, Tokyo, Japan).

Techniques: Cell Culture, Control, Expressing

Journal: PLoS ONE

Article Title: EGF Receptor Exposed to Oxidative Stress Acquires Abnormal Phosphorylation and Aberrant Activated Conformation That Impairs Canonical Dimerization

doi: 10.1371/journal.pone.0023240

Figure Lengend Snippet: Serum-starved A549 cells were incubated (or not) with 1 µM AG1478 or 5 µM PP1 for 30 min. Then, the cells were treated for 30 min. with 100 ng/ml EGF or 1 U/ml GO, as indicated. EGFR was IPed from cell lysates, resolved by SDS-PAGE and IBed for total receptor, total tyrosine phosphorylation (p-EGFR) and specific Tyr-residue phosphorylation level (Y845, Y1068, Y1086, and Y1173). Protein aliquots of the cell lysates were also directly IBed for total and Y416 phosphorylated (active) c-Src (p-Src).

Article Snippet: Primary Abs used in this study for the IBs were: α2232 (αEGFR, Cell Signaling, 1∶1000), αc-Src (Santa Cruz Biotech, 1∶2000), α tyrosine Y416 phosphorylated (p-) c-Src (Cell Signaling, 1∶2000), αp-Y20 (Santa Cruz Biotech, 1∶3000), αp-Y1173 EGFR (Santa Cruz Biotech, 1∶1000), αp-Y1086, αp-Y1068 and αp-Y845 EGFR (Cell Signaling, 1∶1000).

Techniques: Incubation, SDS Page

Journal: PLoS ONE

Article Title: EGF Receptor Exposed to Oxidative Stress Acquires Abnormal Phosphorylation and Aberrant Activated Conformation That Impairs Canonical Dimerization

doi: 10.1371/journal.pone.0023240

Figure Lengend Snippet: A549 cells were incubated (or not) with 5 µM PP1 for 45 min. and then treated (or not) for 15 min. with 100 ng/ml EGF or 30 min. 1 U/ml GO. A . EGFR was IPed from total cell lysates with the mAb 528 and IBed for Y416 phosphorylated c-Src (p-Src) and for total EGFR, as indicated. B . c-Src was IPed from total cell lysates and IBed for total c-Src and EGFR, as indicated.

Article Snippet: Primary Abs used in this study for the IBs were: α2232 (αEGFR, Cell Signaling, 1∶1000), αc-Src (Santa Cruz Biotech, 1∶2000), α tyrosine Y416 phosphorylated (p-) c-Src (Cell Signaling, 1∶2000), αp-Y20 (Santa Cruz Biotech, 1∶3000), αp-Y1173 EGFR (Santa Cruz Biotech, 1∶1000), αp-Y1086, αp-Y1068 and αp-Y845 EGFR (Cell Signaling, 1∶1000).

Techniques: Incubation

Journal: PLoS ONE

Article Title: EGF Receptor Exposed to Oxidative Stress Acquires Abnormal Phosphorylation and Aberrant Activated Conformation That Impairs Canonical Dimerization

doi: 10.1371/journal.pone.0023240

Figure Lengend Snippet: A . Serum-starved A549 cells were treated, or not, with 100 ng/ml EGF for 15 min. or 2% (w/v) MβCD for 1 h and cell lysates were IBed for total and Tyr-phosphorylated EGFR. B and C . Cells were treated with EGF as in A or with 1 U/ml GO for 30 min. in the absence or presence of 2 mM MβCD-cholesterol complex (CC), prepared as described in “ ”. Cell lysates were separated by SDS-PAGE and IBed for total and Tyr-phosphorylated EGFR ( B ) or total and Y416 phosphorylated Src ( C ).

Article Snippet: Primary Abs used in this study for the IBs were: α2232 (αEGFR, Cell Signaling, 1∶1000), αc-Src (Santa Cruz Biotech, 1∶2000), α tyrosine Y416 phosphorylated (p-) c-Src (Cell Signaling, 1∶2000), αp-Y20 (Santa Cruz Biotech, 1∶3000), αp-Y1173 EGFR (Santa Cruz Biotech, 1∶1000), αp-Y1086, αp-Y1068 and αp-Y845 EGFR (Cell Signaling, 1∶1000).

Techniques: SDS Page

Journal: PLoS ONE

Article Title: EGF Receptor Exposed to Oxidative Stress Acquires Abnormal Phosphorylation and Aberrant Activated Conformation That Impairs Canonical Dimerization

doi: 10.1371/journal.pone.0023240

Figure Lengend Snippet: A549 cells were seeded on cover-glasses, serum starved for 1 h and treated (or not) with 1 U/ml GO as indicated. After treatments, ceramide, EGFR phosphorylated on Y1173 and Y416 phosphorylated c-Src were localized in situ by IF as indicated in “ ”; nuclei were stained by DAPI.

Article Snippet: Primary Abs used in this study for the IBs were: α2232 (αEGFR, Cell Signaling, 1∶1000), αc-Src (Santa Cruz Biotech, 1∶2000), α tyrosine Y416 phosphorylated (p-) c-Src (Cell Signaling, 1∶2000), αp-Y20 (Santa Cruz Biotech, 1∶3000), αp-Y1173 EGFR (Santa Cruz Biotech, 1∶1000), αp-Y1086, αp-Y1068 and αp-Y845 EGFR (Cell Signaling, 1∶1000).

Techniques: In Situ, Staining

Journal: PLoS ONE

Article Title: EGF Receptor Exposed to Oxidative Stress Acquires Abnormal Phosphorylation and Aberrant Activated Conformation That Impairs Canonical Dimerization

doi: 10.1371/journal.pone.0023240

Figure Lengend Snippet: A549 cells were seeded on cover-glasses, serum starved for 1 h and treated (or not) with 1 U/ml GO as indicated. After treatments, ceramide ( A and B ), EGFR phosphorylated on Y1173 ( A ) and Y416 phosphorylated c-Src ( B ) were localized in situ by IF as indicated in “ ”; nuclei were stained by DAPI. White arrows indicate regions where p-Y1173 EGFR and ceramide ( A ) or p-Y416 c-Src (p-Src) and ceramide ( B ) co-localized under ox-stress (GO). Z-stack sections of cells have been discriminated by confocal microscopy: the panels show the merge of all of the Z-stack sections.

Article Snippet: Primary Abs used in this study for the IBs were: α2232 (αEGFR, Cell Signaling, 1∶1000), αc-Src (Santa Cruz Biotech, 1∶2000), α tyrosine Y416 phosphorylated (p-) c-Src (Cell Signaling, 1∶2000), αp-Y20 (Santa Cruz Biotech, 1∶3000), αp-Y1173 EGFR (Santa Cruz Biotech, 1∶1000), αp-Y1086, αp-Y1068 and αp-Y845 EGFR (Cell Signaling, 1∶1000).

Techniques: In Situ, Staining, Confocal Microscopy