Journal:

Article Title: Rap2B-Dependent Stimulation of Phospholipase C-? by Epidermal Growth Factor Receptor Mediated by c-Src Phosphorylation of RasGRP3

doi: 10.1128/MCB.24.11.4664-4676.2004

Figure Lengend Snippet: EGF receptor-mediated tyrosine phosphorylation of RasGRP3 by c-Src. (A) HEK-293 cells were transfected with HA-tagged RasGRP1 or RasGRP3 (25 μg of DNA each). After 48 h, the cells were incubated for 30 min without (−) or with (+) 10 μM AG1478 and then stimulated for 5 min without (−) or with (+) 100 ng of EGF/ml as indicated. WB, Western blot; α-HA, anti-HA. (B) HEK-293 cells were transfected with RasGRP3 (25 μg of DNA) alone or with kinase-deficient K298M c-Src or kinase-deficient K457A Pyk2 (75 μg of DNA each). After 48 h, the cells were incubated for 30 min without (−) or with (+) 10 μM PP2 and then stimulated for 5 min without (−) or with (+) 100 ng of EGF/ml as indicated. After cell lysis, the HA-tagged RasGRPs were immunoprecipitated, separated by SDS-PAGE, and probed with antiphosphotyrosine (α-PY) antibodies. Expression of K298M c-Src and K457A Pyk2 was confirmed by immunoblot experiments with specific antibodies (data not shown). (C) Purified GST-tagged RasGRP3 was subjected to in vitro kinase assays without (−) or with (+) recombinant c-Src in the presence of [γ-32P]ATP or unlabeled ATP. Proteins were separated by SDS-PAGE, and RasGRP3 was detected by Coomassie staining, by autoradiography (32P), or by immunoblotting with an antiphosphotyrosine antibody (α-PY). The results are representative of three to five experiments.

Article Snippet: AG1478, BAPTA/AM, Gö 6976, PP2, and recombinant c-Src were from Calbiochem-Merck Biosciences.

Techniques: Transfection, Incubation, Western Blot, Lysis, Immunoprecipitation, SDS Page, Expressing, Purification, In Vitro, Recombinant, Staining, Autoradiography

Journal: Biomolecules

Article Title: Growth Factor Dependent Regulation of Centrosome Function and Genomic Instability by HuR

doi: 10.3390/biom5010263

Figure Lengend Snippet: Recombinant HuR protein phosphorylation by Abl-1 and SRC kinases, in vitro . The phosphorylation of recombinant HuR protein (200 ng) by recombinant ABL1, Src and EGFR kinases detected with pY antibody in Western blots. Note the significant HuR phosphorylation by Abl-1 and SRC kinases.

Article Snippet: The recombinant HuR protein phosphorylation by recombinant c-Abl, EGFR, c-SRC kinases (ProQinase GmbH, Freiburg, Germany) and (New England Biolabs Inc., Beverly, MA, USA) were performed at 30 °C for 40–60 min in Kinase buffer (Cell Signaling Technology, Danvers, MA, USA) supplemented with Mg-ATP.

Techniques: Recombinant, Phospho-proteomics, In Vitro, Western Blot