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. Author manuscript; available in PMC: 2010 Dec 1.
Published in final edited form as: Nature. 2010 Jun 24;465(7301):1084–1088. doi: 10.1038/nature09128

Figure 2. SphK1 is required for TNF-α-induced Lys 63-linked polyubiquitination of RIP1.

Figure 2

a, A7 cells transfected with siControl or siSphK1 were stimulated with TNF-α (10 ng/ml). Proteins were immunoblotted with anti-RIP1 or anti-p65 antibodies. b, Lysates were immunoprecipitated with anti-RIP1 antibody and analyzed with anti-ubiquitin antibody. c, Lysates from cells transfected with HA-Ub and stimulated with TNF-α were immunoprecipitated with anti-RIP1 antibody and analyzed with HA antibody. d, Lysates were immunoprecipitated with anti-RIP1 antibody and proteins analyzed with Lys 63-specific polyubiquitin antibody. e, HEK 293 cells transfected with siControl or siSphK1 were transfected with Flag-TRAF2 and stimulated with TNF-α. Proteins were pulled down with anti-Flag beads and analyzed with anti-TRAF2 antibody.