2.1. cDNA Expression Constructs
Full length human IFNAR2 was amplified from a full length cDNA clone (Invitrogen, Carlsbad, CA), digested with SalI and cloned into the XhoI-digested retrovirus vector, GFPRV. A c-myc tag sequence was inserted by PCR into the extracellular domain within 6 amino acids downstream of the leader peptide. The c-myc tag was inserted with primers: 5-TCTGAAGAAGATCTGTACACAGATGAATCTT GCACTTTC, and 5-AATAAGTTTTTGTTCATCAGGCGAATCATATGAAATACC.
For GST pulldown assays, partial cDNA fragments encoding the COOH-domains of mIFNAR1, mIFNAR2, hIFNAR1, hIFNAR2, and the N-domain of mSTAT4 were amplified by PCR from full length cDNA-containing vectors. The human IFNAR1 cytoplasmic domain and murine STAT4 N-domain PCR products were digested with BamHI and ligated into the BamHI site of the prokaryotic expression vector pGEX-2TK (Amersham Biosciences, Piscataway, NJ). The human IFNAR2, murine IFNAR1 and IFNAR2 cytoplasmic domain PCR products were digested with EcoR1 and ligated into the EcoR1 site within pGEX-2TK.
For yeast-2 hybrid analysis, cDNA fragments encoding the COOH-domain of human IFNAR1 were amplified by PCR, digested with EcoR1 and BamHI and cloned as GAL4 fusion proteins into the EcoR1/BamHI-digested Bait vector pGADT7 (BD-Clontech, Mountain View, CA). The human IFNAR2 PCR product was digested with EcoRI and BamHI and ligated into the EcoRI/BamHI site within pGADT7. The N-domain of mSTAT4 PCR product was digested with BamHI and XhoI and ligated into the BamHI/SalI site within pGADT7. Additionally, cDNA fragments encoding the N-domain, coiled-coil domain, DNA binding domain, linker domain, SH2 domain, and the COOH-terminal domain of murine STAT4 were amplified by PCR, digested with EcoRI/XhoI or BamHI/XhoI and cloned into the EcoRI/SalI or BamHI/SalI sites within the yeast expression vector pFBL23 (Beranger et al., 1997
). This vector allows for the expression of N-terminal fusion proteins with the DNA binding domain of the LexA transcription factor, and these constructs are referred to as Target vectors throughout this study.
2.2. GST Pulldown Assays
GST fusion proteins were expressed in E. coli strain BL21 CodonPlus-RIL (Stratagene, La Jolla, CA). Expression of recombinant proteins was induced by the addition of 0.6 mM IPTG to 300 ml cultures that achieved log-phase growth at 37°C. Upon induction, the cultures were incubated for 3 h at 25°C to maintain stability of recombinant proteins. Cells were disrupted by sonication in 15 ml of lysis buffer (50 mM Tris-Cl (pH 8.0), 1 mM EDTA, 150 mM NaCl, 0.1% Triton-X 100, 5 mM benzamadine, 5 μg/ml leupeptin, 1 mM phenyl methyl sulfonyl fluoride (PMSF), and 0.2 mg/ml lysozyme), and the supernatant was subsequently incubated with glutathione-Sepharose (Amersham Biosciences) at 4°C for 1 h. Following extensive washes, recombinant protein-bound microspheres were incubated in 0.5 ml of a glutathione-sepharose pre-cleared cell lysate derived from 2fTGH cells expressing murine STAT4 as described below. Binding reactions were incubated for 1 h at 4°C followed by extensive washes in wash buffer (10 mM Tris-Cl (pH 7.6), 1 mM EDTA, 150 mM NaCl, 0.5% Triton-X 100). Bound proteins were resolved by 7% SDS-PAGE and transferred to PVDF membranes. STAT4 was detected by immunoblotting with anti-STAT4 antibody (SC-486, Santa Cruz Biotechnology, Santa Cruz, CA) or with a STAT4-N-domain antibody (SC-7959, Santa Cruz) and Gt-anti-Rb-HRP secondary antibody (Jackson Immunoresearch, West Grove, PA) followed by chemilluminescence detection.
2.3. Yeast 2-Hybrid Analysis
S. cerevisiae strains L40 (MATa, trp1, leu2, his3, LYS::lexA-HIS3, URA3::lexA-lacZ) and AMR70 (MATα, trp1, lys2, leu2, his3, URA3::(lexAop)8-lacZ) were propagated to log phase growth in YPD medium (2% peptone, 1% yeast extract, and 2% dextrose). Expression vectors pFBL23 and pGADT7 were introduced into yeast strains AMR70 and L40, respectively, by Li acetate-mediated transformation, and plated onto selective medium. Single transformants of AMR70-pFBL23 were plated on dropout medium (BD-Clontech) lacking tryptophan and uracil. L40-pGADT7 transformants were plated on dropout medium lacking leucine, lysine and uracil at 30°C for 24–48 h. AMR70 and L40 single transformants were mated in 0.5 ml YPD cultures at 30°C for 20 h followed by replica plating on dropout medium lacking tryptophan, uracil, leucine, lysine and in the absence or presence of histidine. Replica mating plates were allowed to grow for 3–5 days at 30°C for detection of specific interactions in the absence of histidine.
2.4. Immunoprecipitation and Western Blotting
For direct immunoprecipitation, cells were lysed in RIPA buffer (150 mM NaCl, 1.0% NP-40, 0.5% (w/v) deoxycholate, 0.1% (w/v) SDS, and 50 mM Tris-Cl (pH 8.0)) and sequential immunoprecipitation was performed with polyclonal anti-STAT1 (SC-346) and anti-STAT4 (SC-486) antibodies. Phosphorylated STAT4 were detected with polyclonal anti-phospho-STAT4 (Tyr 693, Zymed Laboratories, South San Francisco, CA) and phosphorylated STAT1 was detected with anti-phospho-STAT1 (Tyr 701, Upstate, Lake Placid, NY) in combination with a polyclonal Gt-anti-Rb-HRP secondary antibody followed by chemilluminescence detection. Blots were subsequently probed with anti-STAT4 (SC-486) and anti-STAT1 (SC-346) to detect relative amounts of STAT immunoprecipitates.
Cell lysates for co-immunoprecipitation assays () were prepared by solubilizing cells in digitonin buffer (50 mM Tris-Cl (pH 8.0), 150 mM NaCl, 0.8% (w/v) digitonin (Sigma-Aldrich, St. Louis, MO), supplemented with 10 mM phenylmethyl sulfonyl fluoride, 5 μg/ml leupeptin, and 5 mM benzamadine). Duplicate cell lysates were immunoprecipitated with anti-STAT4 (NB34) and anti-c-myc (clone 9E10, Santa Cruz Biotechnology) antibodies. Immunoprecipitates were washed extensively in lysis buffer prior to SDS-PAGE. Immunoblotting was performed as described above.
Fig. 4 STAT4 interacts with the hIFNAR2 cytoplasmic domain in live cells. Cell lysates were prepared from U5A cells (IFNAR2-deficient) expressing STAT4 (lanes 1–3) and either a full-length c-myc-tagged hIFNAR2 (myc-hR2-FL, lane2) or a receptor molecule (more ...)