Journal: Oncogene

Article Title: The Shb signalling scaffold binds to and regulates constitutive signals from the Epstein-Barr virus LMP2A membrane protein.

doi: 10.1038/sj.onc.1210298

Figure Lengend Snippet: Figure 2 Shb and the Shb SH2 domain associate with LMP2A in a phosphotyrosine-dependent manner. (a and b) The association of Shb with LMP2A as a function of LMP2A tyrosine phosphorylation. (c and d) The association of the Shb SH2 domain with LMP2A and its dependence on tyrosine phosphorylation of the LMP2A signalling domain. (a) Immunoprecipitates of LMP2A were probed with the LMP2A mAb 14B7 to show expression of LMP2A in the cell lines used (top panel). Immunoprecipitates were carried out with 14B7 mAb covalently coupled to Sepharose CL-4B to reduce IgH chains in the subsequent immunoblots. The next panel shows a reprobe with antibody to phosphotyrosine demonstrating that the wt LMP2A is constitutively tyrosine phosphorylated, whereas the LMP2A-CD38 fusion protein in the 3Tm cell line is not (Winberg et al., 2000). In the third panel, immunoprecipitates of Shb were probed with LMP2A antibody. The constitutively tyrosine-phosphorylated wt LMP2A in the 1C2 and C4 cell lines (lanes 2 and 3) was brought down by the Shb antibody, whereas the N-terminal domain of LMP2A in the chimeric CD38-LMP2A protein was not detected (lane 4). The last panel shows control immunoprecipitates with normal rabbit serum (NRS). IgH denotes the immunoglobulin heavy chain. (b) The CD38 exodomain of the 3Tm fusion protein was crosslinked either with IB-4 mAb (three top panels) or with OX34 mAb against the rat CD2 exodomain (bottom panel). Since CD2 is absent from 3Tm cells, the OX34 antibody is an irrelevant antibody. After adding the primary crosslinking IB4 and the secondary F(ab)02 antibody, the cells were either lysed directly (0 min) or incubated 10 min at 371C before lysis. The primary crosslinking antibody was here utilized as primary antibody for the immunoprecipitation. Thus, Protein G Sepharose was added to the lysates. In the top panel, the immunoprecipitates were probed with the 14B7 mAb against LMP2A. The second panel shows a reprobe with phosphotyrosine antibody (pY), whereas the third and bottom panels were probed with rabbit antiserum against Shb. (c) Glutathione-Sepharose-bound GST protein and a Glutathione-Sepharose- immobilized GST-Shb SH2 fusion protein were compared for their ability to interact with wt LMP2A in cell lysates. C4 and 1C2 are independent HEK 293 cell clones expressing the wt LMP2A. (d) the 3Tm cell line and 293P vector control cells were subjected to crosslinking with the IB4 mAb as described in Materials and methods. Pull-down assays using the GST-Shb SH2 fusion protein were immunoblotted with anti-phosphotyrosine antibody (upper panel) and reprobed with the 14B7 mAb against LMP2A (lower panel). Trace bands of LMP2A at the 0 min time point in (d) likely reflect that a low level of LMP2A tyrosine phosphorylation takes place during handling of the cells before lysis.

Article Snippet: The anti-rat CD2 hybridoma OX34 was purchased from American Type Culture Collection and was purified as described (Matskova et al., 2001).

Techniques: Phospho-proteomics, Expressing, Western Blot, Control, Incubation, Lysis, Immunoprecipitation, Clone Assay, Plasmid Preparation

Journal: Development (Cambridge, England)

Article Title: Synchronous and symmetric migration of Drosophila caudal visceral mesoderm cells requires dual input by two FGF ligands.

doi: 10.1242/dev.068791

Figure Lengend Snippet: Fig. 3. FGF signaling supports CVM cell survival. Embryos are oriented with anterior towards the left and dorsal upwards. (A-C)Embryos containing the croc-lacZ reporter gene stained using anti-bgal (red) and anti-FasIII (green) to identify CVM and TVM cells, respectively. Wild-type (stage 13; A), Df(2R)BSC25 (stage 13; B) and Df(2R)BSC25 embryos expressing p35 via G447.Gal4 driver (stage 13; C) are depicted. (C)Expression of the anti-apoptotic protein p35 rescues the morphology of CVM cells and allows cells to migrate anteriorly, but cells (asterisk) fail to reach the anterior-most position of the TVM (white line). (D-F)Cell death is observed in FGF mutants using the TUNEL assay. Wild-type embryo (i.e. G447.Gal4 UAS-CD2) of stage 13 (D) and Df(2R)BSC25 G447.Gal4 UAS-CD2 embryos of stage 12 (E) and stage 13 (F) stained with anti-CD2 to detect CVM cells (red) and apoptosis using the TUNEL assay (green).

Article Snippet: The following antibodies were used in the study: rabbit anti-b-gal antibody (1:400; Molecular Probes), mouse anti-Fas III antibody (1:10; Developmental Studies Hybridoma Bank), mouse anti-CD2 antibody (1:300; Serotec), mouse anti-bio (1:500; Roche) and sheep antidig (1:500; Roche).

Techniques: Staining, Expressing, TUNEL Assay

Journal: The Journal of Experimental Medicine

Article Title: The Runx1 Transcription Factor Inhibits the Differentiation of Naive CD4 + T Cells into the Th2 Lineage by Repressing GATA3 Expression

doi: 10.1084/jem.20021200

Figure Lengend Snippet: Effect of cotransduction of GATA3 and Runx1 on Th2 cell differentiation. (A) Naive CD4 + T cells were isolated from the spleens of wild-type and GATA3 -transgenic mice, TCR-stimulated, infected by retroviruses carrying pMX or pMX-Runx1, and cultured in the presence of IL-2. The GFP + population was sorted and reactivated via TCR. The cytokines secreted into the culture supernatant were measured by ELISA. The values obtained for the pMX-Runx1–infected cells are presented as percent inhibition of secretion, taking the values observed for the pMX-infected cells to be 100%. (B) Naive CD4 + T cells were isolated from the spleens of IL-4R (−/−) mice, TCR-stimulated, coinfected by pMX-GFP and pMX-rat CD2 retroviruses as indicated, and cultured in the presence of IL-2. The rat CD2 + population was sorted, TCR-stimulated, and processed for flow cytometrical analysis of intracellular IL-4. Data are representative of two independent experiments.

Article Snippet: Anti-rat CD2 PE (LFA-2) was purchased from Cedarlane.

Techniques: Cell Differentiation, Isolation, Transgenic Assay, Infection, Cell Culture, Enzyme-linked Immunosorbent Assay, Inhibition

Journal:

Article Title: Effects of cyclosporin A and a rapamycin derivative (SAR943) on chronic allergic inflammation in sensitized rats

doi: 10.1046/j.1365-2567.2003.01672.x

Figure Lengend Snippet: Effects of cyclosporin A (CsA) and SAR943 on CD2+ (a), CD4+ (b) and CD8+ (c) T-cell recruitment to the airways following repeated challenge with allergen. There was a significant increase in the number of (a) CD2+ (**P < 0·01) T cells and (b) CD4+ (*P < 0·05) T cells around the airways following repeated allergen exposure of rats compared with repeated saline exposure. Neither CsA nor SAR943 attenuated the recruitment of CD2+ T cells to the airways as compared with the vehicle-treated group (a). SAR943, but not CsA, attenuated the recruitment of CD4+ T cells to the airways, as compared with the vehicle-treated group (#P < 0·05) (b). Neither CsA nor SAR943 altered CD8+ T-cell recruitment compared with vehicle-treated rats. Data are expressed as mean values ± standard error of the mean (SEM).

Article Snippet: For staining CD2 + , CD4 + or CD8 + T lymphocytes in tissue sections, the sections were incubated with mouse anti-rat CD2, CD4 or CD8 mAb (pan T-cell markers; PharMingen, Cambridge Bioscience, Cambridge, UK).

Techniques: