Figure 3.
Figure 3. Distinct signaling pathways are activated by internal and external VEGF-KDR autocrine loops: NF-κB. (A) Nuclear (N) protein extracts from HEL and HL-60 cells, untreated or treated with the external VEGF blocker Ab 4.6.1 or the internal KDR inhibitor (KDRi) for 24 hours, and probed using Western blotting against the p65 and p50 NF-κB subunits. Note the decrease in nuclear p65 and c-rel levels in cells treated with the Ab 4.6.1, while those exposed to KDRi show a clear decrease in p65 levels but sustained c-rel expression. The results are consistent for the 2 cell lines. These results are representative of 3 independent experiments, and were repeated at least 3 times. (B) Cytosolic (C) protein extracts from HEL and HL-60 cells, untreated or treated with the external VEGF blocker Ab 4.6.1 or the internal KDR inhibitor (KDRi) for 24 hours, and probed using Western blotting against the p65 and p50 NF-κB subunits. Note the decrease in cytosolic p65 and c-rel levels in cells treated with the Ab 4.6.1, while those exposed to KDRi also show a clear decrease in p65 levels but sustained c-rel expression. p50 subunit levels did not change in any of the conditions tested, and in either cell line. All the results were consistent for the 2 cell lines. These results are representative of 3 independent experiments, and were repeated at least 3 times. (C) Note the decrease in DNA (NF-κB) binding activity of nuclear protein extracts from HEL or HL-60 cells treated with the 4.6.1 Ab, while those treated with KDRi show little effect (after 12 or 24 hours). The results shown are representative of 3 independent experiments, and were repeated 3 times. (D) Supershift assay of nuclear protein extracts from HEL or HL-60 cells, coincubated with p65-, p50-, or c-rel–specific antibodies, to demonstrate the identity of the NF-κB subunits seen throughout the experiments described earlier.

Distinct signaling pathways are activated by internal and external VEGF-KDR autocrine loops: NF-κB. (A) Nuclear (N) protein extracts from HEL and HL-60 cells, untreated or treated with the external VEGF blocker Ab 4.6.1 or the internal KDR inhibitor (KDRi) for 24 hours, and probed using Western blotting against the p65 and p50 NF-κB subunits. Note the decrease in nuclear p65 and c-rel levels in cells treated with the Ab 4.6.1, while those exposed to KDRi show a clear decrease in p65 levels but sustained c-rel expression. The results are consistent for the 2 cell lines. These results are representative of 3 independent experiments, and were repeated at least 3 times. (B) Cytosolic (C) protein extracts from HEL and HL-60 cells, untreated or treated with the external VEGF blocker Ab 4.6.1 or the internal KDR inhibitor (KDRi) for 24 hours, and probed using Western blotting against the p65 and p50 NF-κB subunits. Note the decrease in cytosolic p65 and c-rel levels in cells treated with the Ab 4.6.1, while those exposed to KDRi also show a clear decrease in p65 levels but sustained c-rel expression. p50 subunit levels did not change in any of the conditions tested, and in either cell line. All the results were consistent for the 2 cell lines. These results are representative of 3 independent experiments, and were repeated at least 3 times. (C) Note the decrease in DNA (NF-κB) binding activity of nuclear protein extracts from HEL or HL-60 cells treated with the 4.6.1 Ab, while those treated with KDRi show little effect (after 12 or 24 hours). The results shown are representative of 3 independent experiments, and were repeated 3 times. (D) Supershift assay of nuclear protein extracts from HEL or HL-60 cells, coincubated with p65-, p50-, or c-rel–specific antibodies, to demonstrate the identity of the NF-κB subunits seen throughout the experiments described earlier.

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