Coro1A is not recruited into the IS and its gene ablation strongly reduces NF-κB responses. (A, B) Confocal microscopy of Coro1A and PKCθ in primary human T cells. A T cell clone (KS140) specific for the tetanus toxin peptide (TT830–843; QYIKANSKFIGITE) and a T cell clone (6396p5.1.2) specific for the measles virus fusion protein peptide (F254–268; GDLLGILESRGIKAR) were used with autologous Epstein–Barr virus (EBV)-transformed B cells as APC. Quantification of Coro1A subcellular localization on 59 synapses is shown as bar graph. (C) CD3+ T cells were isolated from either wild-type or Coro1a knockout mice. After 2 hour resting ex vivo the cells were stimulated with soluble anti-CD3/CD28 and cross-linking anti-hamster IgG antibodies or PDBu for 5 and 15 minutes. Whole cell lysates (supplemented with phosphatase inhibitor) were subjected to SDS-Page and immunoblotting against phosphorylated IκBα, actin and Coro1A. (D) CD3+ T cells were isolated and stimulated as described in (C), but the stimulation time was increased to 8 hours. Nuclear extracts were prepared and analysed by electromobility shift assays (EMSA) for NF-κB binding to DNA. Experiments were repeated at least two times, with similar results.