Fig. 6.
Fig. 6. Constitutive activation of STAT3 in HD cell lines is independent of IL-6, the IL-6R gp80, or gp130. / (A) The cell lines L1236 (lanes 1-5) and L428 (lanes 6-10) were cultured as described in “Materials and methods.” The cells were incubated for 3 days with antibodies against IL-6 (lanes 1 and 6; α-IL-6 B-F6 nonneutralizing, lanes 2 and 7; α-IL-6 B-E8 neutralizing), gp130 (lanes 3 and 8; α-gp130 B-R3), gp80 (lane 4 and 9; α-gp80 B-R6), or gp80 together with gp130 (lanes 5 and 10). Nuclear extracts (5 μg) of each probe were analyzed in a gel retardation experiment using radiolabeled oligonucleotides containing the consensus STAT1 and 3 binding sites as described in the legend to Figure 1. The position of STAT3 and STAT1 homodimers and heterodimers is indicated by arrows. (B) Nuclear extracts from HepG2 cells incubated with IL-6 (250 U/mL) in the absence of an antibody (lane 1), in the presence of neutralizing α-IL-6 (B-E8) antibodies (lane 2), in the presence of nonneutralizing α-IL-6 (B-F6) antibodies (lane 3) and antagonistic α-gp130 (B-R3) antibodies (lane 4) were analyzed for STAT activation. The ability to neutralize IL-6 in the supernatant of L428 cells producing 260 pg/mL IL-6 within 24 hours was tested by incubating L428 cells with neutralizing α-IL-6 (B-E8) or nonneutralizing α-IL-6 (B-F6) antibodies. The L428 supernatants were taken after 20 hours. The corresponding supernatants were incubated with HepG2 cells (lanes 5-7). Nuclear extracts (5 μg) of each probe were analyzed in a gel retardation experiment using radiolabeled oligonucleotides containing the consensus STAT3 binding sites as described in the legend to Figure 1. The position of STAT3/3 homodimers is indicated by an arrow. (C) Western blot analysis of phosphorylated STAT3. Nuclear extracts (20 μg) of each cell line were analyzed in a 10% SDS-polyacrylamide gel, transferred to a nylon membrane, incubated with an antibody specific for phosphorylated STAT3, and visualized by ECL. (D) Western blot analysis of unphosphorylated STAT3. The same nylon membrane as in panel C was stripped and reprobed with an antibody specific for STAT3 and the protein was visualized by ECL.

Constitutive activation of STAT3 in HD cell lines is independent of IL-6, the IL-6R gp80, or gp130.

(A) The cell lines L1236 (lanes 1-5) and L428 (lanes 6-10) were cultured as described in “Materials and methods.” The cells were incubated for 3 days with antibodies against IL-6 (lanes 1 and 6; α-IL-6 B-F6 nonneutralizing, lanes 2 and 7; α-IL-6 B-E8 neutralizing), gp130 (lanes 3 and 8; α-gp130 B-R3), gp80 (lane 4 and 9; α-gp80 B-R6), or gp80 together with gp130 (lanes 5 and 10). Nuclear extracts (5 μg) of each probe were analyzed in a gel retardation experiment using radiolabeled oligonucleotides containing the consensus STAT1 and 3 binding sites as described in the legend to Figure 1. The position of STAT3 and STAT1 homodimers and heterodimers is indicated by arrows. (B) Nuclear extracts from HepG2 cells incubated with IL-6 (250 U/mL) in the absence of an antibody (lane 1), in the presence of neutralizing α-IL-6 (B-E8) antibodies (lane 2), in the presence of nonneutralizing α-IL-6 (B-F6) antibodies (lane 3) and antagonistic α-gp130 (B-R3) antibodies (lane 4) were analyzed for STAT activation. The ability to neutralize IL-6 in the supernatant of L428 cells producing 260 pg/mL IL-6 within 24 hours was tested by incubating L428 cells with neutralizing α-IL-6 (B-E8) or nonneutralizing α-IL-6 (B-F6) antibodies. The L428 supernatants were taken after 20 hours. The corresponding supernatants were incubated with HepG2 cells (lanes 5-7). Nuclear extracts (5 μg) of each probe were analyzed in a gel retardation experiment using radiolabeled oligonucleotides containing the consensus STAT3 binding sites as described in the legend to Figure 1. The position of STAT3/3 homodimers is indicated by an arrow. (C) Western blot analysis of phosphorylated STAT3. Nuclear extracts (20 μg) of each cell line were analyzed in a 10% SDS-polyacrylamide gel, transferred to a nylon membrane, incubated with an antibody specific for phosphorylated STAT3, and visualized by ECL. (D) Western blot analysis of unphosphorylated STAT3. The same nylon membrane as in panel C was stripped and reprobed with an antibody specific for STAT3 and the protein was visualized by ECL.

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