Figure 3.

Map3k1 Regulates Jnk Activation in iNKT Cells

(A) iNKT cells were isolated (four mice per experiment) and stimulated by TCR crosslinking with antibodies over a 240-min time course as indicated. Cell lysates were made and analyzed by IB with the indicated antibodies. Arrowhead indicates phospho-p38 and asterisk a non-specific band.

(B) IPA network diagram of TCR signaling to show the presence of the Mekk1 PHD substrate Carma within this pathway.

(C) In vitro ubiquitination assays using Mekk1 PHD, Mekk1 mPHD, Ube2N:Ube2V1, E1, Ub, and Carma1. Reactions were performed as indicated and analyzed by IB as indicated. A fraction of the ubiquitination reactions was taken pre-incubation, boiled, analyzed by IB as shown, and indicated as input.

(D) HEK293 cells were transfected with the indicated constructs. To detect in vivo ubiquitination, lysates were made under denaturing conditions for IP (Gallagher et al., 2007) and IB performed as indicated. Lysates were also made under non-denaturing conditions as a loading control and IB performed with the indicated antibodies.

(E) iNKT cells were isolated (four mice per experiment) and stimulated by TCR crosslinking with antibodies over a 60-min time course. To detect in vivo ubiquitination, lysates were made under denaturing conditions for IP (Gallagher et al., 2007) and IB performed as indicated. Lysates were also made under non-denaturing conditions as a loading control and IB performed with the indicated antibodies.