src (n16) antibody  (Santa Cruz Biotechnology)

Journal: Oncogene

Article Title: LMP1 signaling pathway activates IRF4 in latent EBV infection and a positive circuit between PI3K and Src is required

doi: 10.1038/onc.2016.380

Figure Lengend Snippet: LMP1 interacts with Src. (a) LMP1 interacts with Src in vitro. 293T cells in 60-mm dishes were transfected with 1 μg HA-LMP1 (or Flag-c-Src) and 1 μg Flag-IRF4 expression plasmids. Cells were collected 48 h post-transfection and then subjected to immunoprecipitation with the Flag antibody clone M2. Beads were washed extensively and then subjected to immunoblotting with the HA antibody clone HA-7. Inputs (5%) were subjected to immunoblotting with the indicated antibodies. (b) Endogenous LMP1 and Src interact in EBV latency. Cell lysates (approximately 1 mg of total proteins in each sample) prepared from JiJoye, IB4 and SavIII cells were subjected to immunoprecipitation with the c-Src antibody clone N16 (or rabbit serum as control). Beads were washed extensively and probed with LMP1 and Src antibodies. Inputs (5%) were also probed with these antibodies. (c) A diagram showing the LMP1 deletion mutant LMP1Δ(12–20). (d) The LMP1 SH3-binding region is not required for Src interaction. 293T cells in 60-mm dishes were transfected with 1 μg LMP1 or LMP1Δ(12–20) and 1 μg Flag-c-Src expression plasmids. Cells were collected 48 h post-transfection and then subjected to immunoprecipitation with the LMP1 antibody clone CS1-4. Beads were washed extensively and then subjected to immunoblotting with the Flag antibody clone M2 and the LMP1 antibody. Inputs (5% of total lysates) were subjected to immunoblotting with indicated antibodies. (e) The LMP1 SH3-binding region is not required for IRF4 activation. The 293 cells in 24-well plates were transfected with 50 ng IRF4 and 50 ng LMP1 (or LMP1Δ(12–20)) expression plasmids, 40 ng pGL3/IFNβ-Luc and 10 ng Renilla. Dual luciferase assay was performed. Results are the averages ± s.e. of duplicates. Representative results from at least three independent experiments are shown. The ability of the vector control to activate the promoter construct was set to 1.

Article Snippet: The lysates were then incubated overnight with 2 μg rabbit Src antibody N16 or LMP1 antibody CS1-4 or normal serum (21st Century Biochemicals Inc., Marlborough, MA, USA).

Techniques: In Vitro, Transfection, Expressing, Immunoprecipitation, Western Blot, Control, Mutagenesis, Binding Assay, Activation Assay, Luciferase, Plasmid Preparation, Construct

Journal: Oncogene

Article Title: LMP1 signaling pathway activates IRF4 in latent EBV infection and a positive circuit between PI3K and Src is required

doi: 10.1038/onc.2016.380

Figure Lengend Snippet: P85 mediates LMP1 and Src interaction. (a) Cell lysates (approximately 1 mg of total proteins in each sample) prepared from JiJoye and IB4 cells were subjected to immunoprecipitation with the LMP1 antibody clone CS1-4 (or mouse serum as control). Beads were washed extensively and probed with P85 and LMP1 antibodies. Inputs (5%) were also probed with these antibodies. (b) LMP1 CTAR1 is responsible for its interaction with Src. 293T cells in 60-mm dishes were transfected with 1 μg 3XMyc-Src and 1 μg Flag-LMP1 or its deletion mutants as indicated. Cells were collected 48 h post-transfection and then subjected to immunoprecipitation with 1.5 μg Myc antibody 9E10 (Roche). Beads were washed extensively and then subjected to immunoblotting with the Flag antibody clone M2 and Myc antibody 9E10. Loading control is Ponceau S staining. Upper panel shows a diagram showing the LMP1 protein structure. (c) Endogenous P85 and Src interact in EBV latency. Cell lysates (approximately 1 mg of total proteins in each sample) prepared from JiJoye, IB4 and SavIII cells were subjected to immunoprecipitation with the c-Src antibody clone N16 (or rabbit serum as control). Beads were washed extensively and probed with P85 and Src antibodies. (d) P85 depletion disrupts the interaction between LMP1 and Src. IB4 cells were infected with retrovirus expressing P85-specific shRNAs (or control). Stable transfectants were selected with 1 μg/ml puromycin for 2 weeks. shRNA expression was induced by 1 μg/ml doxycycline for 3 days. Knockdown efficiency is shown in the lower panel. Cells were then harvested and subjected to IP with the Src antibody clone N16, and then probed with the LMP1 antibody clone CS1-4. Representative results from at least three independent experiments are shown.

Article Snippet: The lysates were then incubated overnight with 2 μg rabbit Src antibody N16 or LMP1 antibody CS1-4 or normal serum (21st Century Biochemicals Inc., Marlborough, MA, USA).

Techniques: Immunoprecipitation, Control, Transfection, Western Blot, Staining, Infection, Expressing, shRNA, Knockdown

Journal:

Article Title: Ubiquitin-mediated degradation of active Src tyrosine kinase

doi:

Figure Lengend Snippet: In vivo ubiquitination of active c-Src. (A) Cos-7 cells were transiently transfected with pCMV4 vector alone (lane 1) or pCMV4 expressing c-Src (lane 2), E378G (lane 3), Myc-tagged ubiquitin (lane 4) alone, or in the combinations indicated (lanes 5–8). For each transfection, 1 mg of lysate was immunoprecipitated with polyclonal antibody (PAb) N16 (anti-Src), separated by SDS/PAGE, transferred to PVDF membrane, and probed with mAb 9E10 (anti-myc). Cells used for lanes 6 and 8 were treated with 50 μM Z-L3VS for 14 hr before harvesting. * indicates a background band. (B) csk−/− cells were treated with either 50 μM Z-L3VS in DMSO or DMSO alone as a negative control. One milligram of lysate was immunoprecipitated with PAb N16 (anti-Src), separated by SDS/PAGE, transferred to PVDF membrane, and probed with mAb Ubi-1 (anti-ubiquitin).

Article Snippet: mAb 327 (anti-Src antibody; Oncogene Science and generous gift of Dr. Thomas), polyclonal antibody PAb N16 (anti-Src antibody; Santa Cruz Biotechnology), polyclonal antibody PAb C11 (anti-actin antibody; Santa Cruz Biotechnology), mAb 9E10 (anti-Myc antibody; Oncogene Science), and mAb Ubi-1 (anti-ubiquitin antibody; Zymed) were used.

Techniques: In Vivo, Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, SDS Page, Negative Control