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. Author manuscript; available in PMC: 2014 Jul 10.
Published in final edited form as: Mol Immunol. 2010 Dec 14;48(4):415–422. doi: 10.1016/j.molimm.2010.09.003

Fig. 1.

Fig. 1

MDA5 is SUMOylated at its C-terminus. (A) MDA5 is SUMO-1 modified. HEK293T cells were transfected with plasmids for Flag-MDA5, HA-SUMO-1 and Myc-Ubc9 by standard calcium phosphate precipitation. Whole cell lysates (WCL) were prepared 48 h post transfection, and immunoprecipitated with anti-Flag M2 antibody. SUMO-conjugated proteins were immunoblotted with anti-SUMO-1 antibody. The whole cell lysates was immunoblotted using anti-Flag as a loading control. (B) Ubc9 is dependent on MDA5 SUMOylation. HEK293T cells were transfected with plasmids for Flag-MDA5, HA-SUMO-1 and Myc-Ubc9 or Ubc9 (C93S) as indicated. Immunoprecipitation was performed exactly as in (A) except that the input was immunoblotted with anti-Flag and anti-Ubc9. (C) The C-terminus of MDA5 is the SUMO-1 acceptor site. HEK293T cells were co-transfected with Flag-tagged full length MDA5, MDA5-N (aa 1–295) or MDA5-C (aa 295–1025) with SUMO-1-HA and Myc-Ubc9 as indicated. SUMOylation of MDA5 was measured exactly as in (A). (D) Endogenous MDA5 is SUMOylated. A549 cells (1 × 107) were transfected with HA-SUMO-1 and Myc-Ubc9 plasmids, or with control plasmids using Lipofectamine 2000. Cell lysates were prepared 48 h after transfection and were immunoprecipitated with anti-MDA5 antibody (Santa Cruz, sc-48031), and detected with anti-MDA5 (ENZO Life Science, ALX-804-863) or anti-SUMO-1 antibody.