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. 2011 Apr 25;286(24):21500–21510. doi: 10.1074/jbc.M110.207951

FIGURE 4.

FIGURE 4.

Up-regulation of ADAM12 by Notch requires an active NF-κB pathway. A, NIH3T3 cells were incubated with CHO-Dll1 or CHO-V cells for 16 h in the absence or presence of MG132, a proteasomal inhibitor. Glycoprotein-enriched cell fractions were analyzed by Western blotting using anti-ADAM12 and anti-Myc antibodies, and β1 Integrin indicates a gel loading control. The nascent form of ADAM12 is indicated by an arrowhead, and the mature form of ADAM12 is shown by an arrow. B, NIH3T3 cells were co-transfected with the 4×CSL luciferase reporter and pRL-TK, an internal control. After 24 h, MG132 or vehicle (DMSO) was added, and the relative luciferase activity was determined 16 h later. The data represent the means ± S.E. from 2 independent measurements; *, p < 0.05. C, NIH3T3, CSL+/+, and CSL−/− cells were transfected with the Igκ2-IFN luciferase reporter, together with pRL-TK. After 18 h, 10 μm DAPT or DMSO was added to transfected cells, and the relative luciferase activity was determined 24 h later. The data represent the means ± S.E. from 4 independent measurements, *, p < 0.05, **, p < 0.01, n.s., non-significant. D, NIH3T3 cells were infected with caNotch1 or control viruses and incubated for 24 h with the indicated concentrations of BMS-345541, an inhibitor of IκB kinase. ADAM12 protein levels were examined by Western blotting after glycoprotein enrichment, and β1 Integrin indicates a gel loading control. NICD1 levels were measured in total cell lysates, and Tubulin indicates a gel loading control. E, NIH3T3 cells were co-cultured with CHO-Dll1 or CHO-V cells for 24 h with the indicated concentrations of BMS-345541. ADAM12 and Dll1-Myc levels were analyzed in glycoprotein-enriched fractions, and β1 Integrin indicates a loading control. The experiments in panels A, D, and E were repeated three times with similar results.