Figure 1

AIP interacts with CARMA1 in overexpression experiments and thereby competes with intramolecular CARMA1 association. (A) Interaction of overexpressed CARMA1 aa 600–946 (PDZ-SH3), CARMA1 aa 600–1147 (C-term) and CARMA1 full length with AIP full length, while CARMA1 aa 1–623 (N-term) is not interacting with AIP. HEK293 cells were co-transfected with FLAG-AIP and different HA-tagged CARMA1 constructs as indicated. After lysis, co-immunoprecipitation (co-IP) was carried out using anti-HA antibody and analysed by Western Blotting. (B) Interaction of overexpressed AIP full length, AIP aa 49–330 (Y2H interactor) and AIP aa 1–130 (PPI domain) with CARMA1 aa 600–946. AIP aa 130–230 (TPR1) and AIP aa 230–330 (TPR2 and 3) are not interacting. Experiment was performed analogous to (A) as anti-FLAG IP. Asterisk indicates migration of IgGs. (C) Schematic summary of the interaction between CARMA1 and AIP fragments. (D) Interaction of HA-CARMA1 aa 932–1147 (GUK) with Strep-FLAG-CARMA1 aa 600–946 (PDZ-SH3). HEK293 cells were co-transfected with both CARMA1 constructs and the binding was analyzed by Western Blotting after Strep-Tactin PD. (E, F) AIP and CARMA1 aa 932–1147 (GUK) bind to CARMA1 aa 600–946 (PDZ-SH3) in a competitive manner. (E) HEK293 cells were co-transfected with Strep-FLAG-CARMA1 aa 600–946, HA-AIP and rising concentrations of HA-CARMA1 aa 932–1147. Strep-Tactin PD was performed as in (D). (F) HEK293 cells were co-transfected and analyzed essentially as in (E), however using constant amounts of HA-CARMA1 aa 932–1148 and rising concentrations of HA-AIP.