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. 2005 Jun;25(11):4716–4726. doi: 10.1128/MCB.25.11.4716-4726.2005

FIG. 8.

FIG. 8.

ASB3 inhibits JNK activation by TNF-R2. (A) 293 cells were cotransfected with TNF-R2 and HA-JNK1 and with pcDNA3, ASB3, or ASB3ΔSB. Forty-eight hours after transfection, whole-cell extracts were prepared and immunoprecipitated with anti-HA (JNK1). Immunoprecipitated HA-JNK1 was analyzed in an in vitro kinase assay with GST-c-Jun as the substrate. The bottom panel shows immunoprecipitated HA-JNK1 blotted with anti-HA. (B) 293 cells were cotransfected with HA-JNK1 and with TRAF-2 and ASB3 (or ASB3ΔSB). The immunoprecipitated HA-JNK1 was analyzed in an in vitro kinase assay with GST-c-Jun as the substrate. The expression levels of TRAF2 in the cell extracts (anti-TRAF2 blotting) are shown in the bottom panel. (C and D) 293 cells were cotransfected with c-Jun-Gal4- and Gal4-responsive luciferase reporters, Renilla luciferase reporter constructs, and pcDNA, mTNF-α, TNF-R2, c-Myc-ASB3, or c-Myc-ASB3ΔSB, as indicated. After 24 h, whole-cell extracts were prepared and split into half for immunoblotting with the indicated antibodies (D) and for a dual-luciferase assay (C).