Journal: PLoS ONE
Article Title: Dynamin2 controls Rap1 activation and integrin clustering in human T lymphocyte adhesion
Figure Lengend Snippet: Chemical inhibition of dynamin2 does not affect integrin affinity regulation. ( A - F ) FACS analysis of the expression of different beta 2 -integrin (CD18) affinity states on the surface of primary human resting CD4 + T lymphocytes in suspension using specific monoclonal antibodies. Cells were either incubated with DMSO as a control or with dynasore to inhibit dynamin2 activity. If indicated, lymphocytes were stimulated with 50ng/ml PMA or 1mM Mn 2+ . Antibodies used were ( A , D , n = 6) KIM127, recognizing intermediate affinity CD18, ( B , E , n = 4) 327C and ( C , F , n = 6) mAb24, both recognizing high affinity CD18. One representative histogram ( A - C ) as well as the relative quantification of surface expression with the stimulated DMSO sample set to 1 ( D - F ) is depicted for each antibody. ( G , H ) Analysis of the static adhesion of primary human resting CD4 + T cells to an ICAM-1-Fc coated surface. If indicated, adhesion was stimulated with KIM185, an antibody mechanically inducing the high affinity conformation of CD18 by binding it on the cellular surface. ( G ) Detailed DIC-images showing representative ICAM-1-Fc coated areas with adherent CD4 + T lymphocytes following incubation with DMSO/dynasore. ( H , n = 4) Quantification of total adherent CD4 + T cells per mm 2 45min after seeding them on an ICAM-1-Fc coated surface. ( I ) Histogram depicting the results of a FACS analysis of the binding of KIM185 antibody to CD18 expressed on the surface of human resting CD4 + T cells following 2h of incubation with DMSO or dynasore. Mean +SEM, **P≤0.01, ***P≤0.001, ns means not significant.
Article Snippet: Chemical inhibitors The following small molecules were used for the chemical inhibition of specific proteins or cellular processes: brefeldin A (B6542, Sigma-Aldrich), chlorpromazine (C8138, Sigma-Aldrich), cytochalasin D (C8273, Sigma-Aldrich), dynasore hydrate (D7693, Sigma-Aldrich), dynole 34–2 (ab120463, Abcam), dynole 31–2 (ab120464, Abcam), Exo1 (#1850, Tocris), latrunculin A (L5163, Sigma-Aldrich), monodansylcadaverine (30432, Sigma-Aldrich).
Techniques: Inhibition, FACS, Expressing, Incubation, Activity Assay, Binding Assay