PKCθ physically interacts with Coro1A. (A) The cartoon depicts interactions identified between deletion mutants of PKCθ and Coro1A by Y2H as well as Co-IP experiments. It could be shown by deletion assays that the WD40 domain of Coro1A and the C2-like domain of PKCθ are sufficient for their interaction. (B) Co-immunoprecipitation analysis of Jurkat T cells transiently transfected with empty vector pEF-neo or RGSH6-tagged Coro1A constructs (wild-type or deletion mutant). Twenty-four hours after transfection, PKCθ-specific immunoprecipitates were immunostained with antibodies recognizing the tag, PKCθ or Coro1A. To control for the specificity of the interaction immunoprecipitation with control IgG (MOCK) was performed. (C) GST-Coro1A pull-down of endogenous PKCθ from mouse CD3+ T cells. WCE = whole cell extract as positive control was used in parallel. (D) Jurkat T cells were transiently transfected with empty vector or RGSH6-tagged wild-type Coro1A together with GFP or PKCθ allelic versions (wild-type, A/E – constitutively active and K/R – dominant negative). Twenty-four hours after transfection, PKCθ-specific antibodies were used for immunoprecipitation and the pulled-down proteins were analysed by immunoblotting the fusion-tag as well as Coro1A and PKCθ-specific antibodies. Experiments were repeated at least three times, with similar results.