![Figure 3. HMGN1 activation of DCs is dependent on TLR4 and MyD88. (A) DCs generated from bone marrow progenitors of C57BL/6 mice were incubated at 5 × 105/ml in medium containing the indicated concentration of HMGN1 for 48 h, and cytokines in the supernatant were measured by cytokine array. Shown is the mean (±SD) of triplicate wells of one experiment representative of three. *, P < 0.05 by Student’s t test when compared with DCs cultured without HMGN1. (B) Mouse bone marrow–derived DCs were treated with 1 µg/ml HMGN1 for 20 or 60 min before they were solubilized in lysis buffer (106 DCs/0.1 ml), and the levels of I-κBα and phosphorylated MAPKs (p44/42, p38, and JNK) in DC lysates were detected by Western blot. After stripping, the membranes were reprobed with GAPDH and the unphosphorylated MAPKs antibodies. The results of one experiment representative of two are shown. (C) Bone marrow–derived DCs from MyD88+/+ and MyD88−/− mice were cultured at 5 × 105/ml without or with 1 µg/ml HMGN1 for 48 h, and cytokines were measured by cytokine array. Shown is the mean (±SD) of three independent experiments. *, P < 0.05 by ANOVA. (D) BM-derived DCs from C3HeN and C3HeJ mice were cultured at 5 × 105/ml without or with 1 µg/ml HMGN1 for 48 h, and cytokines were measured by cytokine array. Shown is the mean (±SD) of three independent experiments. *, P < 0.05 by ANOVA. (E) HMGN1 was incubated with complexes of the extracellular domain of TLR4 (TLR4 ECD) and MD2 for 30 min followed by immunoprecipitation (IP) with rabbit anti-HMGN1 (top) or mouse anti-MD2 (bottom) IgG. The immunoprecipitated proteins were separated by SDS-PAGE and Western blotted (WB) with anti-polyhistidine (top) or anti-HMGN1 (bottom) antibody. Shown are the results of one experiment representative of two. (F) 0.5 nM FLAG–TLR4ECD–MD2 was incubated with 0.8 nM [3H]LOS–CD14 in the absence or presence of endotoxin or 150 nM HMGN1. The FLAG–TLR4ECD–MD2–[3H]LOS in the reaction mixture was captured by anti-FLAG–coated beads. The radioactivity of captured [3H]LOS and uncaptured [3H]LOS was measured by scintillation spectrometry. The binding of [3H]LOS to TLR4ECD–MD2 was shown as the percentage of [3H]LOS captured. The results of one experiment representative of two are shown. (G) TLR4ECD was incubated with MD2–[3H]LOS in the absence or presence of HMGN1, and the reaction mixture was analyzed by Sephacryl HR S300 chromatography. Shown are the results of one experiment representative of two.](https://cdn.rupress.org/rup/content_public/journal/jem/209/1/10.1084_jem.20101354/5/jem_20101354_fig3.jpeg?Expires=1767657454&Signature=P0MuRW~EFREqEKlkTKxRDVLRO25S4Xt8-b7uDWtJ8q8MXJPSdeGaiiCDqiztDDV3L82gFeCUdRtj0HL9EUrXQf~1RZWULu5THPO~gfCek~wIDJypb89wivPWAvGoGEh9Gbp6i8i4vGkMdIUCsPW70yIwp9rgeZRzIjPNKeYw4oRifSQ5a0JLuGHON4v5DhN6WGOiqnVoTUfEsBNV52rYi3yPtnpb13q4EnX5ycom9HoNlkJue74rCyEdoAqVPN4OsgJQyORqdtDJCNCPXoinWvkAI9WFd58~146kIDxhwOYbTKp~79gQ6PS~DR03YnxZ2mXgyVcI9RM-Ggq6QyGtDg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
HMGN1 activation of DCs is dependent on TLR4 and MyD88. (A) DCs generated from bone marrow progenitors of C57BL/6 mice were incubated at 5 × 105/ml in medium containing the indicated concentration of HMGN1 for 48 h, and cytokines in the supernatant were measured by cytokine array. Shown is the mean (±SD) of triplicate wells of one experiment representative of three. *, P < 0.05 by Student’s t test when compared with DCs cultured without HMGN1. (B) Mouse bone marrow–derived DCs were treated with 1 µg/ml HMGN1 for 20 or 60 min before they were solubilized in lysis buffer (106 DCs/0.1 ml), and the levels of I-κBα and phosphorylated MAPKs (p44/42, p38, and JNK) in DC lysates were detected by Western blot. After stripping, the membranes were reprobed with GAPDH and the unphosphorylated MAPKs antibodies. The results of one experiment representative of two are shown. (C) Bone marrow–derived DCs from MyD88+/+ and MyD88−/− mice were cultured at 5 × 105/ml without or with 1 µg/ml HMGN1 for 48 h, and cytokines were measured by cytokine array. Shown is the mean (±SD) of three independent experiments. *, P < 0.05 by ANOVA. (D) BM-derived DCs from C3HeN and C3HeJ mice were cultured at 5 × 105/ml without or with 1 µg/ml HMGN1 for 48 h, and cytokines were measured by cytokine array. Shown is the mean (±SD) of three independent experiments. *, P < 0.05 by ANOVA. (E) HMGN1 was incubated with complexes of the extracellular domain of TLR4 (TLR4 ECD) and MD2 for 30 min followed by immunoprecipitation (IP) with rabbit anti-HMGN1 (top) or mouse anti-MD2 (bottom) IgG. The immunoprecipitated proteins were separated by SDS-PAGE and Western blotted (WB) with anti-polyhistidine (top) or anti-HMGN1 (bottom) antibody. Shown are the results of one experiment representative of two. (F) 0.5 nM FLAG–TLR4ECD–MD2 was incubated with 0.8 nM [3H]LOS–CD14 in the absence or presence of endotoxin or 150 nM HMGN1. The FLAG–TLR4ECD–MD2–[3H]LOS in the reaction mixture was captured by anti-FLAG–coated beads. The radioactivity of captured [3H]LOS and uncaptured [3H]LOS was measured by scintillation spectrometry. The binding of [3H]LOS to TLR4ECD–MD2 was shown as the percentage of [3H]LOS captured. The results of one experiment representative of two are shown. (G) TLR4ECD was incubated with MD2–[3H]LOS in the absence or presence of HMGN1, and the reaction mixture was analyzed by Sephacryl HR S300 chromatography. Shown are the results of one experiment representative of two.
