Figure 4. Membrane-associated and... | Rockefeller University Press

Figure 4.

$Figure 4. Membrane-associated and especially soluble TNF are dramatically increased in IECs of LPS-challenged IKKβ(EE)IEC mice. (a) Lysates of small intestinal enterocytes were prepared when indicated after LPS injection and analyzed for TNF expression by immunoblotting (transmembrane, 26 kD; soluble, 17 kD). Representative of three experiments. (b) Frozen sections of WT or IKKβ(EE)IEC jejuna prepared 2 h after LPS injection were stained with anti-TNF antibody and visualized by immunofluorescence. Representative of three experiments. (c) Lysates of small intestinal enterocytes (75 µg protein) of indicated genotypes were prepared when indicated after LPS injection and analyzed for TNF content by ELISA. Results are means ± SEM (n = 4). *, P < 0.05 versus WT. Data are representative of four independent experiments. (d) RNAs from small intestinal enterocytes of indicated genotypes were prepared when indicated after LPS injection and analyzed in triplicates by Q-RT-PCR for TNF mRNA and normalized to Hprt mRNA. *, P < 0.05 versus WT; #, P < 0.05 versus other time points in IKKβ(EE)IEC mice. Results are means ± SEM (n = 4). Data are representative of four independent experiments. (e and f) LPS administration results in only a mild increase of p65 nuclear translocation and DNA binding in IKKβ(EE)IEC enterocytes. (e) WT and IKKβ(EE)IEC enterocytes were isolated 30 min after LPS injection and separated into cytosolic (C) and nuclear (N) fractions that were analyzed for their content of the indicated proteins by immunoblotting. Representative of two experiments. (f) Nuclear extracts of WT and IKKβ(EE)IEC enterocytes isolated 30 min after LPS injection were analyzed for p65 DNA binding by ELISA. Results are means ± SEM (n = 3). *, P < 0.05 versus control. Data are representative of two independent experiments. (g and h) Low LPS dose administration induces TNF protein production by Tg enterocytes without a further increase in its mRNA amounts. (g) Lysates of small intestinal enterocytes (75 µg protein) of indicated genotypes prepared when indicated after low LPS dose (0.2 mg/kg) injection were analyzed for TNF content by ELISA. *, P < 0.05 versus WT. Results are means ± SEM (n = 3). Data are representative of three independent experiments. (h) RNAs from small intestinal IECs prepared when indicated after low LPS dose (0.2 mg/kg) injection were analyzed in triplicates by Q-RT-PCR for TNF mRNA amounts and normalized to Hprt mRNA. Results are means ± SEM (n = 3). *, P < 0.05 versus WT. Data are representative of three independent experiments.$

Membrane-associated and especially soluble TNF are dramatically increased in IECs of LPS-challenged IKKβ(EE)^IEC mice. (a) Lysates of small intestinal enterocytes were prepared when indicated after LPS injection and analyzed for TNF expression by immunoblotting (transmembrane, 26 kD; soluble, 17 kD). Representative of three experiments. (b) Frozen sections of WT or IKKβ(EE)^IEC jejuna prepared 2 h after LPS injection were stained with anti-TNF antibody and visualized by immunofluorescence. Representative of three experiments. (c) Lysates of small intestinal enterocytes (75 µg protein) of indicated genotypes were prepared when indicated after LPS injection and analyzed for TNF content by ELISA. Results are means ± SEM (n = 4). *, P < 0.05 versus WT. Data are representative of four independent experiments. (d) RNAs from small intestinal enterocytes of indicated genotypes were prepared when indicated after LPS injection and analyzed in triplicates by Q-RT-PCR for TNF mRNA and normalized to Hprt mRNA. *, P < 0.05 versus WT; #, P < 0.05 versus other time points in IKKβ(EE)^IEC mice. Results are means ± SEM (n = 4). Data are representative of four independent experiments. (e and f) LPS administration results in only a mild increase of p65 nuclear translocation and DNA binding in IKKβ(EE)^IEC enterocytes. (e) WT and IKKβ(EE)^IEC enterocytes were isolated 30 min after LPS injection and separated into cytosolic (C) and nuclear (N) fractions that were analyzed for their content of the indicated proteins by immunoblotting. Representative of two experiments. (f) Nuclear extracts of WT and IKKβ(EE)^IEC enterocytes isolated 30 min after LPS injection were analyzed for p65 DNA binding by ELISA. Results are means ± SEM (n = 3). *, P < 0.05 versus control. Data are representative of two independent experiments. (g and h) Low LPS dose administration induces TNF protein production by Tg enterocytes without a further increase in its mRNA amounts. (g) Lysates of small intestinal enterocytes (75 µg protein) of indicated genotypes prepared when indicated after low LPS dose (0.2 mg/kg) injection were analyzed for TNF content by ELISA. *, P < 0.05 versus WT. Results are means ± SEM (n = 3). Data are representative of three independent experiments. (h) RNAs from small intestinal IECs prepared when indicated after low LPS dose (0.2 mg/kg) injection were analyzed in triplicates by Q-RT-PCR for TNF mRNA amounts and normalized to Hprt mRNA. Results are means ± SEM (n = 3). *, P < 0.05 versus WT. Data are representative of three independent experiments.

This Feature Is Available To Subscribers Only