Figure 5.

IGSF4 associates with the TCR ζ-chain through the TM domain and boosts TCR-mediated signal transduction. (A, top) J-GFP or J-IGSF4_GFP cells were incubated for 30 min with or without anti-CD3/28. Cell lysates were immunoprecipitated with anti-GFP (top left) or anti–TCR ζ-chain (top right) and then immunoblotted with antibodies against the indicated molecules (ζ-chain, Lck, Zap70, or IGSF4). (middle) HEK293T cells were cotransfected with WT-IGSF4_GFP and TCR ζ_RFP, and then immunoprecipitation (IP) and immunoblotting (IB) were performed as in the top left blots. Confocal images show surface localization of IGSF4_GFP and TCR ζ_RFP. The boxed area (yellow) is represented as zoomed images in the right and bottom micrographs. Bar, 10 µm. (bottom left) J-GFP or J-IGSF4_GFP cell pellets were lysed, immunoprecipitated with anti-GFP, and then immunoblotted with antibodies against the indicated molecules (TCR α and β, CD3γ, CD3δ, CD3ε, TCR ζ, and GFP). Total lysates were also immunoblotted with the same antibodies indicated above. The data are representative of two independent experiments. (bottom right) Immunoprecipitation and immunoblotting of Jurkat T cells expressing GFP, WT-IGSF4_GFP, or the indicated mutants (ΔCT, ΔEXT, CD43TM, and ΔEXT/CD43TM) as in A (top left). The data are representative of at least three independent experiments. EV, empty vector; IG4, IGSF4. (B) Jurkat T cells expressing GFP, WT-IGSF4_GFP, or mutant IGSF4 (ΔCT, ΔEXT, and ΔEXT/CD43TM) were stimulated with plate-bound anti-CD3 for the indicated time. The cells lysates were immunoblotted by using antibodies against phosphorylated (p) and total (t) ζ-chain. The data are representative of three independent experiments. (C) J-GFP or J-IGSF4_GFP cells were stimulated with plate-bound anti-CD3 for the indicated time. The cell lysates were immunoblotted for the phosphorylated and total forms of Lck, Zap70, ERK, and p38 kinase. The data are representative of at least three independent experiments. Molecular mass (M) is indicated in kilodaltons.