Fig. 4

GMAP210 silencing inhibits activation of T lymphocytes. a Immunoblot analysis of signalosomes prepared from Jurkat cells expressing control (C) or GMAP210 specific ShRNA (3, 8) activated for 0, or 10 min with magnetic beads coated with mAb to CD3ε and to CD28 (above blots). Proteins attached to the beads were purified by magnetic sorting after freezing and thawing of the cells without detergent (anti-CD3/CD28). Presence of the different proteins in the corresponding cell lysates are shown in “input” lanes. b Quantification of PLCγ, SLP-76, LAT, and VAMP7 band intensities at 10 min of activation and normalized on CD3ζ intensity band. c and e Enzyme-linked immunosorbent assay of IL-2 in supernatants of Jurkat cells (c) or IFN-γ in human primary CD4+ T cells (e) expressing control (circle) or GMAP210-specific ShRNA (triangle) and activated for 6 h by Raji B cells pulsed with various concentrations (horizontal axis) of SEE (c, Jurkat T cells) or anti-CD3 in the presence of 10 μg/mL of anti-CD28 (e, human primary T cells). d Quantitative PCR analysis of IL-2 in Jurkat cells activated as in c). f Enzyme-linked immunosorbent assay of IFN-γ in supernatants of human primary CD4+ T cells expressing control (ShC) or GMAP210-specific ShRNA (Sh3 and Sh8) and activated for 6 h with a combination of PMA+ionomycin that bypasses LAT-signaling. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: non-significant (paired parametric t-tests, one tail). Mean is represented by horizontal lines in b and each experiment is represented by one color. Data represent between three and five experiments in a, b, c, and e, and three experiments in d and f