Fig. 2.
Fig. 2. LILRE/S1-S3 oligoprecipitations of nuclear extracts from THP-1 cells followed by immunoblot analysis. / (A) Oligoprecipitation by LILRE (lanes 2, 5, and 8) and S1-S3 (lanes 3, 6, and 9) oligonucleotides of nuclear extracts isolated from unstimulated (lanes 1-3), IL-6–stimulated (lanes 4-6), and IFN-γ–stimulated (lanes 7-9) THP-1 cells. Subsequent immunoblot analysis was performed on isolated proteins by using STAT1 antibodies (G16930). Lanes 1, 4, and 7 show control precipitations using only streptavidin-coated sepharose beads. (B) LILRE/S1-S3 oligoprecipitation of nuclear extracts isolated from IL-6–stimulated (lanes 1-3) and IFN-γ–stimulated (lanes 4-6) THP-1 cells, followed by immunoblot analysis using phosphotyrosine-STAT3 (Tyr705) antibodies (no. 9131). Lanes 1 and 4 show control precipitations using beads without coupled oligonucleotides. Additional LILRE oligoprecipitation assays were performed on nuclear extracts isolated from IL-6–stimulated THP-1 cells, followed by immunoblot analysis using STAT3 (F2) antibodies. Lane 7 shows a control precipitation using streptavidin sepharose beads without biotinylated LILRE oligonucleotides. (C) Oligoprecipitation by LILRE (lanes 2, 5, 8) and LILRE/competitive LILRE oligonucleotides (lanes 3, 6, 9) of nuclear extracts from unstimulated (lanes 1-3), IL-6–stimulated (lanes 4-6), and IFN-γ–stimulated (lanes 7-9) THP-1 cells. LILRE-bound proteins were analyzed by Western blot using phosphotyrosine (pY) STAT1 (Y701) antibodies. (D) Competitive oligoprecipitation assay on nuclear extracts from IL-6–stimulated (lanes 1-4) and IFN-γ–stimulated (lanes 5-8) THP-1 cells, followed by immunoblot analysis using pY-STAT3 antibodies. Precipitations were performed using biotin-labeled LILRE oligonucleotides bound to streptavidin sepharose beads (lanes 2-4; 6-8). Excess amounts (12 μg) of double-stranded LILRE oligonucleotides (lanes 3, 7) and TRE oligonucleotides (lanes 4, 8) were added to LILRE-coupled streptavidin beads. Lanes 1 and 5 show control precipitations using uncoupled streptavidin sepharose beads. (E) LILRE/S1-S3 oligoprecipitation of nuclear extracts isolated from IL-6–stimulated (lanes 1-3) and IFN-γ–stimulated (lanes 4-6) THP-1 cells, followed by immunoblot analysis using phosphotyrosine antibodies (PY100). Lanes 1 and 4 show control precipitations using uncoupled streptavidin sepharose beads. Figures represent the results of 3 independent experiments (n = 3). MW indicates molecular weight.

LILRE/S1-S3 oligoprecipitations of nuclear extracts from THP-1 cells followed by immunoblot analysis.

(A) Oligoprecipitation by LILRE (lanes 2, 5, and 8) and S1-S3 (lanes 3, 6, and 9) oligonucleotides of nuclear extracts isolated from unstimulated (lanes 1-3), IL-6–stimulated (lanes 4-6), and IFN-γ–stimulated (lanes 7-9) THP-1 cells. Subsequent immunoblot analysis was performed on isolated proteins by using STAT1 antibodies (G16930). Lanes 1, 4, and 7 show control precipitations using only streptavidin-coated sepharose beads. (B) LILRE/S1-S3 oligoprecipitation of nuclear extracts isolated from IL-6–stimulated (lanes 1-3) and IFN-γ–stimulated (lanes 4-6) THP-1 cells, followed by immunoblot analysis using phosphotyrosine-STAT3 (Tyr705) antibodies (no. 9131). Lanes 1 and 4 show control precipitations using beads without coupled oligonucleotides. Additional LILRE oligoprecipitation assays were performed on nuclear extracts isolated from IL-6–stimulated THP-1 cells, followed by immunoblot analysis using STAT3 (F2) antibodies. Lane 7 shows a control precipitation using streptavidin sepharose beads without biotinylated LILRE oligonucleotides. (C) Oligoprecipitation by LILRE (lanes 2, 5, 8) and LILRE/competitive LILRE oligonucleotides (lanes 3, 6, 9) of nuclear extracts from unstimulated (lanes 1-3), IL-6–stimulated (lanes 4-6), and IFN-γ–stimulated (lanes 7-9) THP-1 cells. LILRE-bound proteins were analyzed by Western blot using phosphotyrosine (pY) STAT1 (Y701) antibodies. (D) Competitive oligoprecipitation assay on nuclear extracts from IL-6–stimulated (lanes 1-4) and IFN-γ–stimulated (lanes 5-8) THP-1 cells, followed by immunoblot analysis using pY-STAT3 antibodies. Precipitations were performed using biotin-labeled LILRE oligonucleotides bound to streptavidin sepharose beads (lanes 2-4; 6-8). Excess amounts (12 μg) of double-stranded LILRE oligonucleotides (lanes 3, 7) and TRE oligonucleotides (lanes 4, 8) were added to LILRE-coupled streptavidin beads. Lanes 1 and 5 show control precipitations using uncoupled streptavidin sepharose beads. (E) LILRE/S1-S3 oligoprecipitation of nuclear extracts isolated from IL-6–stimulated (lanes 1-3) and IFN-γ–stimulated (lanes 4-6) THP-1 cells, followed by immunoblot analysis using phosphotyrosine antibodies (PY100). Lanes 1 and 4 show control precipitations using uncoupled streptavidin sepharose beads. Figures represent the results of 3 independent experiments (n = 3). MW indicates molecular weight.

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