Ramnarain et al.

Supplementary Figure Legends

Figure 1

A, the relative levels of PDGFR in RIP1 knockout mouse embryo fibroblasts compared to wild type cells. Cell lysates from early passage primary MEFs were subjected to Western blot analysis with PDGFR specific antibodies in the upper panel. The blot was stripped and re-probed with STAT3 to show protein loading control (lower panel). B. Reconstitution of RIP1 into RIP1-/- MEFs results in lowering of PDGFR levels. An adenovirus expressing RIP1 under the control of a Tet-off system was infected into MEFs for 48hours followed by Western blotting. Cells exposed to tetracycline do not express RIP1. C. the relative levels of IGF-IR in RIP1 knockout mouse embryo fibroblasts compared to wild type cells. D. levels of IGF-IR in reconstituted cells. E. the relative levels of FGFR3 in RIP1 knockout mouse embryo fibroblasts compared to wild type cells. F. levels of FGFR3 in reconstituted cells.

Figure 2

A. IKKγ/NEMO fails to inhibit the EGFR promoter (pER1-LUC). In this experiment 293 cells were transfected with pER1-LUC along with empty vector (0) or Myc tagged IKKγ/NEMO as shown followed by luciferase assays. B. The same concentration of IKKγ/NEMO completely inhibits TNFα induced activation of a NF-B-Luc promoter demonstrating functional activity of overexpressed IKKγ/NEMO. TNFα was used at a concentration of 50ng/ml for 6 hours. A renilla luciferase construct was used an internal control. Error bars represent SEM of triplicate determinations. The Western blots show expression of Myc tagged IKKγ/NEMO or protein loading (ERK2).

Figure 3

Legend is included with the figure.

Figure 4

Demonstrates expression of FLAG-tagged RIP1 wild type or mutants in 293 cells. This Western blotwas conducted in parallel with the reporter assay shown in Figure 5 E and probed with a FLAG antibody (from Cell Signaling Technology). No signal is detected in the vector transfected lane. DDD: deletion of death domain, DKD: deletion of kinase domain, DID: deletion of intermediate domain. There is a robust expression of the DDD mutant but it fails to suppress Sp1-LUC activity as seen in Figure 5E.