Journal: Molecular Biology of the Cell

Article Title: CD47 plays a critical role in T-cell recruitment by regulation of LFA-1 and VLA-4 integrin adhesive functions

doi: 10.1091/mbc.e13-01-0063

Figure Lengend Snippet: FIGURE 4: Human Jurkat T-cell integrin expression and adhesion to HUVEC monolayers in an in vitro flow model. (A) Jurkat CD47+ clone E6 and CD47− (null) clone JINB8 express similar levels of LFA-1 and VLA-4 integrins. Data are representative of three separate experiments. (B) Jurkat CD47− T-cells were drawn across the TNF-α–activated HUVECs at various estimated shear stress levels as described in Materials and Methods. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 for indicated comparisons (Student’s t test). (C) Both CD47+ and CD47− Jurkat T-cell adhesion (under shear stress of 0.76 dynes/cm2) to TNF-α–activated HUVECs is strongly dependent on VLA-4 integrins. The reduction in adhesion in CD47+ cells with blockade of LFA-1 is not observed in CD47− cells, suggesting that LFA-1–dependent adhesion requires CD47. Data are mean ± SEM of three experiments. *p ≤ 0.05, **p ≤ 0.01 vs. CD47+ with no mAb in medium; #p < 0.05, media CD47− vs. anti-VLA-4 mAb–treated CD47− cells (Student’s t test).

Article Snippet: The β2 integrin was detected by Western blot with goat anti-human β2 integrin polyclonal antibody (R&D Systems, Minneapolis, MN).

Techniques: Expressing, In Vitro, Shear

Journal: Molecular Biology of the Cell

Article Title: CD47 plays a critical role in T-cell recruitment by regulation of LFA-1 and VLA-4 integrin adhesive functions

doi: 10.1091/mbc.e13-01-0063

Figure Lengend Snippet: FIGURE 6: Mn2+ or Mg2+/EGTA fails to induce strong LFA-1 and β1 high-affinity conformation expression or binding of soluble ICAM-1-Fc chimera in CD47− (null) Jurkat T-cells. (A–D) Mn2+ or Mg2+/EGTA–induced expression of LFA-1 extended and open “activated” conformations of LFA-1 were detected by the reporter mAb KIM127 (A) and mAb24 (B). Results are normalized to isotype-matched, control nonbinding mAb. Expression of these active conformations of integrins by 0.5 mM Mn2+ or 10 mM Mg2+/1 mM EGTA treatments was significantly reduced in CD47− Jurkat T-cells. (C) Total levels of β2 and β1 integrins detected with TS1/18 and LIA1/2.1 mAb, respectively, did not change after Mn2+ or Mg2+/EGTA stimulation. No change was detected in CD47 (data not shown). (D) High-affinity β1 integrins induced by Mn2+ or Mg2+/ EGTA buffer were measured by mAb HUTS21. (E) CD47− T-cells exhibited little binding of soluble ICAM-1 as compared with CD47+ T-cells induced by Mn2+ and Mg2+/EGTA treatments. Results are normalized to incubation buffer alone. Data are mean ± SEM, n = 3. *p ≤ 0.05, **p ≤ 0.01 (Student’s t test). (F) Mn2+ activates solubilized β2 integrins from CD47+ (lane 3) and CD47-null (lane 6) Jurkat T-cells. Lysates of Jurkat cells were subjected to immunoprecipitation with anti-β2 integrin mAb 24 (lanes 2, 3 and 5, 6) in the absence (–) or presence (+) of 1.0 mM Mn2+, followed by SDS–PAGE and immunoblotting with anti-β2 integrin polyclonal Ab (R&D Systems). Immunoprecipitation with mAb TS1/18 (lanes 1 and 4) served as a positive control to ensure the presence of β2 integrin in the lysate.

Article Snippet: The β2 integrin was detected by Western blot with goat anti-human β2 integrin polyclonal antibody (R&D Systems, Minneapolis, MN).

Techniques: Expressing, Binding Assay, Control, Incubation, Immunoprecipitation, SDS Page, Western Blot, Positive Control

Journal: Molecular Biology of the Cell

Article Title: CD47 plays a critical role in T-cell recruitment by regulation of LFA-1 and VLA-4 integrin adhesive functions

doi: 10.1091/mbc.e13-01-0063

Figure Lengend Snippet: FIGURE 7: CD47 and β2 integrin interact on the cellular membrane of Jurkat T-cells. (A) Representation of interacting fraction τm by pseudocolor images of the FLIM-FRET analysis of the interaction between β2 integrins with CD47 in unstimulated conditions and upon Mg2+/ EGTA activation, and the interaction between activated β2 integrin detected by mAb 24 and

Article Snippet: The β2 integrin was detected by Western blot with goat anti-human β2 integrin polyclonal antibody (R&D Systems, Minneapolis, MN).

Techniques: Membrane, Activation Assay