A. In a screening strategy designed to identify the MAP3Ks that bind to RIP2, HA-tagged-MEKK2, MEKK3 and MEKK4 were co-transfected into 293 cells with Omni-tagged RIP2. After immunoprecipitation, Western blotting was performed using the indicated antibodies.
B. To determine if RIP2 and MEKK4 co-localized in the cell, CFP-RIP2 and YFP-MEKK4 fusion constructs were generated. 293 cells were transfected, and confocal microscopy was performed. In the basal, unstimulated state, RIP2 and MEKK4 co-localized. Multiple views with merging of the images are shown.
C. To determine if endogenous RIP2 and MEKK4 bound one another and if this complex persists in response to NOD2 activation (via MDP exposure), RAW 264.7 cells were stimulated with 20 μg/mL MDP for 0, 30, 60 or 90 minutes. Cells were lysed and subjected to immunoprecipitation with an anti-MEKK4 antibody or an anti-RIP2 antibody. Western blotting was performed after extensive washing of the immunoprecipitate. As a control for MDP activation, lysates were Western blotted using anti-phospho-IκBα and anti-total IκBα. Endogenous MEKK4 binds to endogenous RIP2 in the unstimulated state. As the NFκB pathway is activated (shown by the anti-phospho-IκBα blot), MEKK4 binding to RIP2 greatly decreases. This pattern is seen in reciprocal co-immunoprecipitations.

A,B. To determine whether NOD2 competes with MEKK4 for RIP2 binding in the unstimulated state, 293s were transfected with Omni-tagged NOD2 and/or HA-tagged MEKK4 or HA-tagged RIP2. NOD2 (panel A) or MEKK4 (panel B) were immunoprecipitated and Western blotting was performed using the indicated antibodies. RIP2 only bound NOD2 when MEKK4 was absent from the experiment.
C, D. A similar experiment to that described in A and B was performed with the exception that in this set of experiments, MDP (10 μg/mL) was transfected into the cell to stimulate NOD2. After MDP exposure, NOD2 (panel C) or MEKK4 (panel D) were immunoprecipitated and Western blotting was performed using the indicated antibodies. In the presence of MDP, NOD2 preferentially bound to RIP2 and could compete with MEKK4 for RIP2 binding.
E, F. To determine if two disease-specific Crohn’s associated NOD2 alleles could compete with MEKK4 for binding to RIP2 in the absence or presence of MDP, two Crohn’s Disease-associated NOD2 alleles (L1007insC and D291N) were transfected with MDP (10 μg/mL) and/or HA-tagged RIP2 and MEKK4 as indicated. The L1007insC is the mouse homolog and instead of causing a truncation mutation, causes a nonsense mutation leading to 41 nucleotide elongation before truncation (see reference 13). MEKK4 was immunoprecipitated and Western blotting was performed using the indicated antibodies. Unlike wt NOD2, neither L1007insC NOD2 nor D291N NOD2 could compete with MEKK4 for binding RIP2 in the presence or absence of MDP.

A. To determine the effect of MEKK4 on RIP2-induced NEMO ubiquitination and to determine the MAP3K specificity of this effect, kinase-dead (denoted as KA — catalytic lysine converted to alanine in each kinase) variants of MEKKs 1, 3 and 4 were transfected into 293s with myc-tagged K399R NEMO, RIP2 and HA-tagged ubiquitin. As a positive control for inhibition of NEMO ubiquitination, kinase dead TAK1 was utilized. After immunoprecipitating NEMO under stringent washing conditions, Western blotting was performed using the indicated antibodies. Kinase-dead MEKK4 specifically and strongly inhibited RIP2-induced NEMO ubiquitination.
B. To determine whether the inhibitory effect of MEKK4 on RIP2-induced NEMO ubiquitination was dependent on the kinase activity of MEKK4, wt MEKK4 or kinase-dead MEKK4 was transfected into 293 cells with myc-tagged K399R NEMO, RIP2 and HA-tagged ubiquitin. After immunoprecipitation, Western blotting was performed. wt MEKK4 inhibited RIP2-induced NEMO ubiquitination with similar efficacy as kinase-dead MEKK4.
C. Mammalian GST-tagged IKKβ was transfected into 293 cells with RIP2 and/or MEKK4. After purification with glutathione-sepharose beads, Western blots were performed. MEKK4 strongly inhibits RIP2-induced IKKβ activation.
D. 293 cells were transfected with IκBα, RIP2 and/or wt MEKK4 or kinase-dead (KA) MEKK4. Lysates were generated and Western blotting was performed. RIP2 strongly induces IκBα phosphorylation and both wt and kinase-dead (KA) MEKK4 inhibit this activation with similar efficacy.
E. Flag-tagged JNK was transfected into 293 cells with RIP2 and/or MEKK4. After immunoprecipitation with the Flag antibody, Western blots were performed with the indicated antibodies. RIP2 strongly activates JNK, and MEKK4 expression has no effect on this activation.
F. Flag-tagged p38 was transfected into 293 cells with RIP2 and/or MEKK4. After immunoprecipitation with the Flag antibody, Western blots were performed. MEKK4 strongly activates p38. RIP2 co-expression had little effect on this activation.

A. RAW 264.7 cells were transduced with individual lentiviruses targeting MEKK4. As a control, an empty lentivirus was used. Western blotting was performed to determine the degree of inhibition of MEKK4 expression. Cell lines MEKK4 shRNA #3 and MEKK4 shRNA #4 showed the greatest loss of expression and were used for subsequent experiments. These cell lines were then exposed to MDP for 0, 30, 60 or 90 minutes. Lysates were generated, and half the lysates were subjected to immunoprecipitation stringent washing conditions (0.25% SDS, 1 M NaCl). Western blots showed that NEMO ubiquitination was increased basally and greatly increased under stimulation in the shRNA#3 and shRNA#4 cell lines but not in the vector only cell line. In agreement with this finding, phospho-IκBα was increased in these cell lines (both basally and with stimulation), indicating higher NFκB activity. In contrast, while JNK signaling was similar between the vector-only cell line and the shRNA#3 and the shRNA#4 cell lines, p38 signaling was greatly decreased with MEKK4 expression was inhibited..
B. To determine if this increased basal NFκB activation resulted in altered cytokine production, cytokine levels were assayed from these cell lines by ELISA. IL-6, IL-10, IL-12, TNFα and G-CSF were all significantly basally upregulated in both MEKK4 knockdown lines. Student’s t-test showed that IL-6, IL-10, TNF and G-CSF increases had a significance of P<0.05 (indicated by *). As these cytokines are all regulated by NFκB, these findings suggest a functional role for the increased NFκB signaling seen in Figure 4A.
C. Expression of two NFκB-regulated proteins was then assayed under either basal conditions or after overnight MDP stimulation. Both RIP2 and A20 were significantly upregulated in the MEKK4 knockdown lines. In contrast, the innate immunity protein, IRAK4, whose expression is not regulated by NFκB activity, was essentially unchanged.