t cell signaling molecule  (Cell Signaling Technology Inc)

Journal: European journal of immunology

Article Title: The SH3 domain of Tec kinase is essential for its targeting to activated CD28 costimulatory molecule.

doi: 10.1002/eji.200324777

Figure Lengend Snippet: Fig. 2. Tec is recruited to CD28 in an SH3-dependent manner. (A) Jurkat cells electroporated with Tec–GFP constructs were incu- bated at 4°C with CD28 mAb (CD28.2) or CD3 mAb (289) for 30 min followed by Texas-red-conjugated goat anti-mouse (Tx-Red) for 30 min. Then, cells were left at 4°C (unstimulated: –) or incubated at 37°C for 10 min (stimulated: xCD28 or xCD3). Different Tec–GFP constructs were tested: the wild-type Tec (Tec-WT, upper panels), a kinase-dead Tec mutant (TecKD, middle panels) and a Tec SH3 mutant (Tec-SH3*, lower panels). Data shown are representative of three independent experiments. (B) T cell hybridoma transfectants with the wild-type (WT) human CD28 molecule or with the human CD28 molecule mutated on the intra- cytoplasmic proline residues (P193A or P181/183A) were stimulated and treated as described in Sect. 4.4. Cells were stained with anti-Tec antibody and examined by confocal microscopy. In all experiments, 20–30 cells were examined per condition. Data shown are representative of two experiments.

Article Snippet: Key words: T cells / Cell signaling / CD28 molecule / Tec kinases / SH3 domain Received 1/12/03 Revised 6/4/04 Accepted 3/5/04 [DOI 10.1002/eji.200324777] Abbreviations: GFP: Green fluorescent protein PH: Pleckstrin-homology PRR: Proline-rich region PTK: Protein tyrosine kinase SEE: Staphylococcus enterotoxin E SH3: Src-homology 3

Techniques: Construct, Incubation, Mutagenesis, Staining, Confocal Microscopy

Journal: European journal of immunology

Article Title: The SH3 domain of Tec kinase is essential for its targeting to activated CD28 costimulatory molecule.

doi: 10.1002/eji.200324777

Figure Lengend Snippet: Fig. 3. The importance of the SH3 domain of Tec and the PRR of CD28 in the regulation of IL-2 production upon CD28 triggering. (A) T cell hybridomas transfected by wild-type (WT) CD28 or CD28 with mutated proline residues (P193A or P181/183A) were stimulated separately by cross-linked CD5 (control), CD3 or CD28 mAb on an FcR+ B cell lymphoma (LK35.2 cells) or were stimulated by a combination of solu- ble CD3 + CD28 mAb. The soluble forms of CD3 or CD28 mAb alone can not induce a strong IL-2 production in these cells. To detect high levels of IL-2 (up to 1 ng/ml), these mAb need to be used in combination or presented at the surface of the LK35.2 cells. Supernatants were collected after 24 h of stimulation. The IL-2 concentration was measured by ELISA and is expressed in ng/ml. Shown are mean values ± SD of four individual points of stimulation, and using differ- ent independent clones for each P/A construct. (B) Jurkat cells were transfected by electroporation with vector alone (pCDNA3) or the indicated Tec constructs: pCDNA3-Tec (Tec) and pCDNA3-TecSH3* (TecSH3*). Cells were cotrans- fected with pIL-2-Luc and g -actinR-Luc. After a 2-h recov- ery, cells were left unstimulated (NS) or treated with CD28 or CD3 mAb in presence of PMA or a combination of CD3 + CD28 mAb for 6 h, then luciferase assays were performed. Data shown are the average ± SD of three independent experiments.

Article Snippet: Key words: T cells / Cell signaling / CD28 molecule / Tec kinases / SH3 domain Received 1/12/03 Revised 6/4/04 Accepted 3/5/04 [DOI 10.1002/eji.200324777] Abbreviations: GFP: Green fluorescent protein PH: Pleckstrin-homology PRR: Proline-rich region PTK: Protein tyrosine kinase SEE: Staphylococcus enterotoxin E SH3: Src-homology 3

Techniques: Transfection, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Clone Assay, Construct, Electroporation, Plasmid Preparation, Luciferase