Journal:

Article Title: Inhibition of Src Family Kinases by a Combinatorial Action of 5?-AMP and Small Heat Shock Proteins, Identified from the Adult Heart

doi:

Figure Lengend Snippet: Demonstration of c-Src inhibitory activity in the cardiocyte lysate. (A) c-Src autophosphorylation and enolase substrate phosphorylation measured in the absence (−) or presence (+) of the cardiocyte lysate (0.1 μg/μl). The positions of c-Src (60 kDa) and enolase (45 kDa) are indicated. (B) Dose response of the effects of cardiac lysate on c-Src activity determined with p34cdc2 synthetic peptide as the substrate. (C) Activity and amount of c-Src in the kinase reaction determined by autoradiography and Western blotting (W. Blot) with c-Src monoclonal antibody GD11, respectively. (D) Cardiocyte lysate was split into <10- and >10-kDa fractions as described in Materials and Methods. Top, c-Src autophosphorylation in the absence or presence of cardiocyte lysate, the <10-kDa fraction, and/or the >10-kDa fraction; bottom, similar experiment performed with c-Src preautophosphorylated with [γ-32P]ATP, where the effect of cardiocyte fractions was measured subsequently with unlabeled ATP. (E) The <10-kDa fraction was treated with buffer, subtilisin, or pronase and then used to study their effects on c-Src autophosphorylation in the absence or presence of the untreated >10-kDa (left) or <10-kDa (right) fraction.

Article Snippet: c-Src-specific monoclonal antibody GD11 was purchased from Upstate Biotechnology, Inc. (Lake Placid, N.Y.), whereas anti-c-Src N-terminus antibody N-16 was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.).

Techniques: Activity Assay, Autoradiography, Western Blot

Journal: PLoS ONE

Article Title: Tyrosine 416 Is Phosphorylated in the Closed, Repressed Conformation of c-Src

doi: 10.1371/journal.pone.0071035

Figure Lengend Snippet: AD293 cells were transfected with the indicated constructs and after 24h analysed by Western Blots as shown. Key: Em, Emerald fluorescent protein; closed, Q528E, P529E, G530I mutant of c-Src; open, Y527F mutant of c-Src; β-gal, β -galactosidase in same vector as c-Src constructs A. The blot was standardized to c-Src levels. Phospho-Y416 was detected with two different antibodies: #1, cat. PK1109 from Calbiochem; #2, cat. 2101 from Cell Signaling. B. The blot was standardized to endogenous STAT3. Blots were performed twice and showed consistent results.

Article Snippet: Antibodies were used at 1∶10,000 dilution for GFP (#A-6455, Invitrogen), 1∶1,000 dilution for STAT3 (#610189, BD Biosciences), 1∶1,000 for STAT3 pY705 (#9131, Cell Signaling), 1∶500 c-Src (#N-16, Santa Cruz Biotechnology), 1∶7,500 for c-Src pY416#1 (#PK1109, Calbiochem), 1∶7,500 for c-Src pY416#2 (#2101, Cell Signaling), 1∶10,000 for α-tubulin (Invitrogen).

Techniques: Transfection, Construct, Western Blot, Mutagenesis, Plasmid Preparation

Journal: PLoS ONE

Article Title: Tyrosine 416 Is Phosphorylated in the Closed, Repressed Conformation of c-Src

doi: 10.1371/journal.pone.0071035

Figure Lengend Snippet: AD293 cells were transfected with the indicated constructs and analysed by Western Blot 24 h post-transfection. Blots were performed at least twice, which showed consistent results; a single representative is shown. Key: KD, kinase dead K293 M mutant; Em, Emerald fluorescent protein; closed, Q528E, P529E, G530I mutant of c-Src; open, Y527F mutant of c-Src; β-gal, β -galactosidase in same vector as c-Src constructs. A. Western Blot standardized for c-Src levels. Phospho-Y416 was detected with two different antibodies: #1, cat. PK1109 from Calbiochem; #2, cat. 2101 from Cell Signaling. B. Western Blot standardized for STAT3 levels.

Article Snippet: Antibodies were used at 1∶10,000 dilution for GFP (#A-6455, Invitrogen), 1∶1,000 dilution for STAT3 (#610189, BD Biosciences), 1∶1,000 for STAT3 pY705 (#9131, Cell Signaling), 1∶500 c-Src (#N-16, Santa Cruz Biotechnology), 1∶7,500 for c-Src pY416#1 (#PK1109, Calbiochem), 1∶7,500 for c-Src pY416#2 (#2101, Cell Signaling), 1∶10,000 for α-tubulin (Invitrogen).

Techniques: Transfection, Construct, Western Blot, Mutagenesis, Plasmid Preparation

Journal: PLoS ONE

Article Title: Tyrosine 416 Is Phosphorylated in the Closed, Repressed Conformation of c-Src

doi: 10.1371/journal.pone.0071035

Figure Lengend Snippet: A. Western Blots of AD293 cells transfected with c-Src variants for 24 h standardized to β-tubulin. C-Src was transfected at different doses by adjusting the proportion of DNA used in transfections from 100% to 1.56% supplemented with a vector expressing non-fluorescent Y66L GFP derivative. Key: closed, Q528E, P529E, G530I mutant of c-Src; open, Y527F mutant of c-Src. Phospho-Y416 was detected with two different antibodies: #1, cat. PK1109 from Calbiochem; #2, cat. 2101 from Cell Signaling. B. Densitometry analysis of the Western Blot data in panel A for STAT3 phospho-Y705 immunoreactivity, c-Src immunoreactivity and c-Src phospho-Y416 immunoreactivity with antibody #1 (cat. PK1109). Blots shown are representative of three independent experiments. Densitometry analysis is for the blots shown.

Article Snippet: Antibodies were used at 1∶10,000 dilution for GFP (#A-6455, Invitrogen), 1∶1,000 dilution for STAT3 (#610189, BD Biosciences), 1∶1,000 for STAT3 pY705 (#9131, Cell Signaling), 1∶500 c-Src (#N-16, Santa Cruz Biotechnology), 1∶7,500 for c-Src pY416#1 (#PK1109, Calbiochem), 1∶7,500 for c-Src pY416#2 (#2101, Cell Signaling), 1∶10,000 for α-tubulin (Invitrogen).

Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing, Mutagenesis

Journal: PLoS ONE

Article Title: Tyrosine 416 Is Phosphorylated in the Closed, Repressed Conformation of c-Src

doi: 10.1371/journal.pone.0071035

Figure Lengend Snippet: A. Western Blots of AD293 cells transfected with c-Src variants for 24 h standardized to β-tubulin. C-Src was transfected at different doses by adjusting the proportion of DNA used in transfections from 100% to 1.56% supplemented with a vector expressing non-fluorescent Y66L GFP derivative. Key: closed, Q528E, P529E, G530I mutant of c-Src; open, Y527F mutant of c-Src. Phospho-Y416 was detected with two different antibodies: #1, cat. PK1109 from Calbiochem; #2, cat. 2101 from Cell Signaling. B. Densitometry analysis of the Western Blot data in panel A for STAT3 phospho-Y705 immunoreactivity, c-Src immunoreactivity and c-Src phospho-Y416 immunoreactivity with antibody #1 (cat. PK1109). Blots shown are representative of three independent experiments. Densitometry analysis is for the blots shown.

Article Snippet: Antibodies were used at 1∶10,000 dilution for GFP (#A-6455, Invitrogen), 1∶1,000 dilution for STAT3 (#610189, BD Biosciences), 1∶1,000 for STAT3 pY705 (#9131, Cell Signaling), 1∶500 c-Src (#N-16, Santa Cruz Biotechnology), 1∶7,500 for c-Src pY416#1 (#PK1109, Calbiochem), 1∶7,500 for c-Src pY416#2 (#2101, Cell Signaling), 1∶10,000 for α-tubulin (Invitrogen).

Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing, Mutagenesis