Figure 6.

Ubp43 interacts with IFNAR2 receptor subunit. (A) 293T cells were transiently transfected with Flag-IFNAR1, HA-IFNAR2, or Flag-IFNGR1 and either GST control or GST-Ubp43. Reciprocal immunoprecipitations (I.P.) were performed using anti-Flag/HA or anti-GST antibodies. Whole-cell lysates (WCL) or immunoprecipitated complexes were subjected to immunoblotting with anti-HA antibodies (top middle panel), anti-Flag (top left & right panels) or anti-GST (bottom panel) antibodies, respectively. (B) Transient co-transfections of 293T cells were performed using HA-tagged IFNAR2 and various deletion mutants of Ubp43 (a.a. positions are indicated in the figure). Ubp43 deletion constructs used for this study are schematically represented in the top panel. WCL or immunoprecipitated (anti-HA) complexes were subjected to immunoblotting with anti-HA antibodies (middle panel) or anti-GST (bottom panel) antibodies, respectively. (C) wt Ubp43 and Ubp43 mutants: D₃₃₁K₃₄₀-AA, R₃₅₀R₃₅₂R₃₅₄-AAA, K₃₆₄-A (positions of a.a. substitutions are graphically shown on the top panel of the figure) were co-expressed with GST-IFNAR2 (a.a. 265–515) in 293T cells. I.P. were performed using anti-HA antibodies followed by immunoblotting with anti-HA antibodies (top) to verify equal loading and anti-GST-antibodies (bottom). (D) U3A cells were transiently co-transfected by the combination of ISRE-driven luciferase reporter plasmid, STAT1, and either vector-control, wt Ubp43, or Ubp43 mutants: D₃₃₁K₃₄₀-AA (DK), R₃₅₀R₃₅₂R₃₅₄-AAA (RRR) and K₃₆₄-A (K). At 24 h post-transfection, cells were either left untreated or treated with hIFN-α for 24 h. Luciferase activities were measured, normalized, and presented as fold increase of relative luciferase activity in IFN treated cells over the untreated controls (average of 4 independent experiments). The error bars indicate the s.d. of the mean.