The StIP1 sequence. Comparison of conceptually translated murine (mu) and human (hu) StIP1 cDNA sequences. WD40 motifs are shaded and labeled 1–12. Dots represent absence and dashes represent identity of corresponding amino acids. Arrows indicate borders of the spliced exon in Δ7 StIP1. Murine StIP1 has a predicted molecular mass of 93 kDa.

Expression pattern of StIP1 transcripts. Expression of StIP1 was evaluated by a reverse transcription–PCR assay (Roche Molecular Biochemicals). RNA was prepared from each of the indicated tissues and amplified with primers from repeat 6 and 8 (Fig. 3A). PCR products corresponding to the full-length (FL) or Δ7 transcripts are indicated. Controls samples (Cntl) were amplified from cDNA templates.

In vitro association of StIP1 with Stat1, Stat3, and Stat5. (A) Schematic diagram of seven GST-StIP1 fusion proteins (GST-StIP1A–GST-StIP1H). (B) GST-StIP1 fusion proteins (lanes 3–6) or GST alone (lane 2) were incubated with HepG2 extracts. Bound proteins were detected with a Stat3-specific antibody (C-20; Santa Cruz Biotechnology). Lane 1 represents 1/20 of the input material for each pulldown. The mobility of Stat3 is indicated. (C) Extracts prepared from unstimulated M1 cells were incubated with indicated GST-StIP1 fusion proteins (lanes 3–6) or GST alone (lane 2) and detected with a Stat3-specific antibody (C-20; Santa Cruz Biotechnology). Lane 1 represents 1/3 of the input material for each pulldown. (D) The filter from C was reprobed with a Stat1-specific antibody (M-22; Santa Cruz Biotechnology). (E) Extracts from unstimulated (lanes 1, 3, and 5) or IL-3-stimulated (lanes 2, 4, and 6) 32D cells were incubated with GST-StIP1A (lanes 3 and 4) or GST alone (lane 5), and then detected with a Stat5b-specific antibody (C-17; Santa Cruz Biotechnology). Input extracts were loaded directly onto the gel in lanes 1 and 2. Lane 2 shows the appearance of a more slowly migrating band corresponding to the IL-3-stimulated tyrosine-phosphorylated isoform of Stat5b (confirmed by antiphosphotyrosine blotting; data not shown). (F) Recombinant Stat3 either inactive/nontyrosine phosphorylated (rStat3; lanes 1–5) or active/tyrosine phosphorylated (rStat3*P; lanes 6–10) was prepared from insect cells infected either with a Stat3 or Stat3 plus Jak1 baculovirus. These recombinant proteins were incubated with the indicated GST-StIP1 fusion proteins (lanes 3–5 and 8–10) or GST alone (lanes 2 and 7) and then detected by Western blotting with a Stat3-specific antibody (C-20; Santa Cruz Biotechnology). The filter then was reprobed with a phosphotyrosine-specific Stat3 antibody (Lower; New England Biolabs).

Association of StIP1 with Stat3 and JAKs. (A) Extracts were prepared from M1 cells either before or after stimulation with IL-6 (200 units/ml). They were immunoprecipitated either with a StIP1 or Stat3 (C-20, Santa Cruz Biotechnology) antibody and subsequently immunoblotted with either a Stat3 (◂; C-20, Santa Cruz Biotechnology), StIP1 (▹), or antiphosphotyrosine (4G10; Upstate Biotechnology) antibodies as indicated. (B) M1 extracts were immunoprecipitated with Jak1 (lanes 1 and 2; Upstate Biotechnology), Jak2 (lanes 3 and 4; Upstate Biotechnology), or Tyk2 (lanes 5 and 6) antibodies and sequentially immunoblotted with StIP1 or the appropriate JAK antibodies as indicated. (C) Extracts from unstimulated (lanes 1–6) and IL-6-stimulated (lanes 7–12) M1 cells were incubated with GST-StIP1 fusion proteins (lanes 3–6 and 9–12) or GST alone (lanes 2 and 8) and bound proteins detected with a Jak1-specific antibody (←; Signal Transduction Laboratories, Lexington, KY).

Stat3, Jak1, and StIP1 do not form a stable trimolecular complex. (A) 293T cells were cotransfected with 5 μg each of RcCMV-Stat3 (25), pMT2T-Jak1^K896R (29), and pCNG0.8 as indicated. Whole-cell extracts (10 μl), prepared after 24 h, were diluted to 500 μl and incubated with 5 μl of α-Stat3 (C-20; Santa Cruz Biotechnology). Precipitates were immunoblotted with either α-StIP1 (Top), α-Jak1 (Middle; Upstate Biotechnology), or α-StIP1 (Bottom). Mobilities of StIP1, Jak1, and Stat3 are indicated in the left margin and M_r in the right margin. (B) Samples, prepared as in A, were immunoprecipitated with 5 μl of α-StIP1 and immunoblotted with α-Jak1 (Upper) or α-StIP1 (Lower). Mobilities of Jak1 and StIP1 are indicated in the left margin and M_r in the right margin. (C) Model of the putative Stat3-StIP1-Jak1 trimolecular complex.

Overexpression of StIP1 mutants blocks cytokine signaling. (A) HepG2 cells were cotransfected with an IRF-1 GAS-driven reporter (32) and either empty vector, full-length StIP1, or pCGN0.8. Extracts were assayed for luciferase activity either before (open bars) or after (filled bars) treatment with IL-6. Data represent results from one experiment done in triplicate, but similar results were obtained in three independent studies. Firefly luciferase activity was normalized to renilla luciferase activity expressed by a control vector, pRLnull (Promega). (B) 293T cells were cotransfected with 1 μg of a Stat3 expression vector (25) and increasing amounts (i.e., 0.5, 1.0, 2.0, and 5.0 μg; lanes 3–6) of a pCGN0.8 expression construct with a His-epitope tag, as indicated. DNA-binding activity was evaluated with an IRF-1 GAS probe either before or after stimulation with IL-6 (200 Units/ml) plus soluble IL-6 receptor (0.5 μg/ml; a generous gift from F. Horn, RWTH, Aachen, Germany; ref. 34). The mobility of STAT DNA-binding complexes is indicated in the right margin. (C–F) HepG2 cells were transfected with 5.0 μg of pCGN0.8 and then evaluated by dual immunostaining with anti-hemagglutinin (C and E; 1° antibody = 12CA5, Roche Molecular Biochemicals; and 2° antibody = goat anti-mouse-Alexa, Molecular Probes), and anti-Stat3 (D and F; 1° antibody = C20, Santa Cruz Biotechnology; and 2° antibody = donkey anti-rabbit-Cy3, Jackson ImmunoResearch), either before (C and D) or after an 8-min stimulation with IL-6 (E and F; 15 ng/ml, PeproTech, Rocky Hill, NJ). (G and H) The localization of endogenous StIP1 in NIH 3T3 cells was evaluated by immunostaining with a murine-specific StIP1 antibody either before or after stimulation with ligand.