Interaction of Stat6 and NF-κB p50 and NF-κB p65 in GST pulldown assays. (A) GST fusion proteins, as indicated, were incubated with nuclear extracts from I.29μ sIgM⁺ B lymphoma cells stimulated with IL-4 for 2 h (lanes c, e, and g) or cells left unstimulated (lanes d, f, and h). Lanes a and b contain the input proteins in the nuclear extracts (10 μg each). The bound proteins were resolved by SDS–10% PAGE, followed by Western blotting with anti-Stat6 (top panel), anti-PU.1 (middle panel), and anti-GST (bottom panel) antibodies sequentially on the same blot. In the bottom panel, the bands below the full-length protein in GST-p65 lanes are shorter forms of GST-p65 fusion proteins. Although the photograph has been cut and spliced, all the lanes are from the same blot, autoradiographed for the same time. This is also true for panels B and C. (B) Nuclear extracts from I.29μ B cells left untreated (lanes c, e, and g) or cultured in the presence of anti-IL-4 antibody (lanes d, f, and h) were incubated with GST fusion proteins as indicated. The bound Stat6 was resolved by SDS–10% PAGE, followed by Western blotting with anti-Stat6 antibody. The result from three-times-longer exposure of the film than that used for panel A is shown. Input Stat6 in the nuclear extracts (10 μg) is shown in lanes a and b. (C) Purified rStat6, untreated or treated with EtBr, was used for binding to GST fusion proteins as indicated in the absence (lanes c, e, g, and i) or presence (lanes d, f, and h) of EtBr. Lanes a and b show the inputs of pretreated rStat6 (5 ng). The bound rStat6 was subjected to SDS–10% PAGE, followed by Western blotting with anti-Stat6 antibody. (D) Nuclear extracts (100 μg) from IL-4-treated (lane a) or untreated (lane b) I.29μ B cells and purified rStat6 (50 ng) (lane c) were immunoprecipitated with anti-Stat6 antibody, and the immunoprecipitates were then subjected to SDS–7.5% PAGE, followed by Western blotting with antiphosphotyrosine antibody (top panel) and anti-Stat6 antibody (bottom panel) sequentially on the same blot.

Requirement for the phosphotyrosines of Stat6 in GST pulldown assays. (A) Nuclear extracts from IL-4-stimulated I.29μ B cells were treated with TCPTP in the presence (lanes c, e, and g) or absence (lanes d, f, and h) of vanadate prior to incubation with the indicated GST fusion proteins. Lanes a and b show the input Stat6 in the pretreated nuclear extracts (10 μg). (B) Purified rStat6 was left untreated (lanes c, e, and g) or pretreated with TCPTP (lanes d, f, and h) prior to incubation with GST fusion proteins. rStat6 was then used for binding to the indicated GST fusion proteins. Lanes a and b show the inputs of pretreated rStat6 (1 ng). The bound Stat6 and rStat6 were resolved by SDS–10% PAGE, followed by Western blotting with anti-Stat6 antibody. The lanes are from the same blot, autoradiographed for the same time.

Interaction of Stat6 and NF-κB p50 in vivo. I.29μ B cells were labeled with [³⁵S]methionine and [³⁵S]cysteine for 3 h. Cells were then treated with LPS for 2 h, followed by stimulation with (lanes 3 and 4) or without (lanes 1 and 2) IL-4 for 30 min. Nuclear extracts were first immunoprecipitated with anti-Stat6 antibodies. Immunoprecipitates were dissolved, and aliquots were reprecipitated with rabbit antiserum against NF-κB p50 or p65. The precipitated proteins were analyzed by SDS–7.5% PAGE and fluorography. The positions of size markers were as indicated (in kilodaltons). Bottom panels: The contents of Stat6 and NF-κB proteins in the nuclear extracts (5 μg each) were analyzed by SDS–10% PAGE, followed by Western blotting with anti-Stat6, anti-p50, or anti-p65 antibody. The samples were analyzed on the same blot and autoradiographed for the same time as the reference samples (lane 1 of each panel) of nuclear extracts (5 μg each) from HEK 293 cells cotransfected with equal amounts of Stat6, NF-κB p50, and NF-κB p65 expression plasmids.

Synergy between Stat6 and NF-κB in activating an IL-4-inducible reporter gene in HEK 293 cells. (A) HEK 293 cells were transfected with one or more expression plasmids for Stat6 or NF-κB as indicated, together with the luciferase reporter plasmid (IL-4 RR)₂-pFL and the control plasmid pfosCAT. Forty hours posttransfection, cells were stimulated with (▪) or without (□) IL-4 for 8 h before being harvested. The luciferase activity was normalized for transfection efficiency by the CAT activity and calculated as a relative value by assigning the activity from unstimulated cells transfected with empty vector alone a value of 1. The data are presented as means plus standard errors of the means (SEMs) from three transfection experiments. (B) HEK 293 cells were transfected with wild-type or other mutated (IL-4 RR)₂-pFL reporter plasmids as pictured, together with expression plasmids for Stat6, NF-κB p50, and NF-κB p65 and the control plasmid pfosCAT. Forty hours posttransfection, cells were stimulated with IL-4 for 8 h before being harvested. The luciferase activity was normalized by the CAT activity and calculated as a percentage relative to the activity from cells transfected with the wild-type reporter plasmid. The data are presented as means plus SEMs from three transfection experiments.

EMSAs demonstrating that Stat6 and NF-κB in nuclear extracts from transfected HEK 293 cells together form an IL-4-inducible complex with the IL-4 RR −124/−79 Cɛ DNA fragment. (A) DNA binding reactions were performed with nuclear extracts (5 μg) from HEK 293 cells transfected with different combinations of expression plasmids as indicated and left untreated (lanes 2, 4, 6, 8, 10, and 12) or stimulated with IL-4 for 15 min (lanes 3, 5, 7, 9, 11, and 13). Lane 1 contains probe alone. Bottom panels: Western blotting analyses of nuclear extracts (5 μg each) corresponding to the samples used in EMSA. The blot was probed with anti-Stat6, anti-NF-κB p65, and anti-NF-κB p50 antibodies sequentially. (B) DNA competition experiments were performed with nuclear extracts (5 μg) from IL-4-treated HEK 293 cells overexpressing Stat6 and NF-κB p50 and p65 (lanes 3 to 6) or Stat6 and NF-κB p50 (lanes 9 to 12). A 100-fold molar excess (2.5 pmol) of unlabeled double-stranded oligonucleotides containing a Stat6 site (lanes 4 and 10), a κB site (lanes 5 and 11), or a size-matched control DNA fragment (lanes 6 and 12) was added to the DNA binding reaction mixture. Binding reactions with nuclear extracts from empty vector-transfected cells are shown in lanes 2 and 8. Lanes 1 and 7 contain probe alone.

Functional synergy between Stat6 and NF-κB in I.29μ B cells. I.29μ B cells were transfected with wild-type or mutated (IL-4 RR)₂-pFL reporter plasmids, as pictured, together with the control plasmid pSV2CAT. Aliquots of the transfected cells were cultured with (▪) or without (□) IL-4 for 15 h before harvesting. The luciferase activity was normalized by the CAT activity and calculated as a percentage relative to the activity from IL-4-treated cells transfected with the wild-type reporter plasmid. The data are presented as means plus standard errors of the means (SEMs) from three transfection experiments.

EMSAs demonstrating that Stat6 and NF-κB in nuclear extracts from I.29μ B cells together form an IL-4-inducible complex with the IL-4 RR −124/−79 Cɛ DNA fragment. Lanes 2 to 3: EMSAs of nuclear extracts from I.29μ B cells untreated (lane 2) or treated with IL-4 (lane 3). Lane 1 contains probe alone. Lanes 4 to 9: DNA competition experiments were performed as in Fig. 5B, except nuclear extracts from untreated (lanes 4, 6, and 8) or IL-4-stimulated (lanes 5, 7, and 9) I.29μ B cells were used in the binding reactions. The unlabeled double-stranded oligonucleotide competitors were added into the binding reaction mixtures as indicated. Lanes 10 to 13: antibody supershift experiments were performed with nuclear extracts from IL-4-treated I.29μ B cells. One microliter of the indicated antibodies was added to the DNA binding reaction.