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. Author manuscript; available in PMC: 2011 Mar 15.
Published in final edited form as: Cancer Res. 2010 Mar 9;70(6):2379–2388. doi: 10.1158/0008-5472.CAN-09-4204

Fig. 4. ANXA1 directly binds to NF-κB p65 subunit in human cancer cells.

Fig. 4

A: Nuclear proteins from BxPC-3 cells treated for 3 h with NO-ASA 20 µM were incubated with κB oligomers immobilized on the wall of the reaction wells of 96-well plates. NF-κB dimers bound to these oligomers were recognized by anti-p65 or anti-p50 antibodies through a color reaction. An anti-ANXA1 mAb (middle column) that did not cross-react with either p50 or p65 generated a positive reaction. Values are mean±SEM (n≥3). *, p<0.001 compared to control. B: Total protein lysates of BxPC-3 pancreatic cancer cells treated with either NO-ASA 30 µM or dexamethasone 4 µM were immunoprecipitated using either an antibody against the p65 subunit of NF-κB or a nonspecific isotypic IgG antibody (control) and immunoblotted (IB) as indicated. C: The same study as in B was performed on HT-29 colon cancer cells giving similar results. D: Left panel: EMSA from BxPC-3 cells treated with NO-ASA up to 30 µM for 3 h. NO-ASA inhibited the binding of NF-κB to the DNA probe. Right panel: In this EMSA, the nuclear protein extract was reacted with an anti-ANXA1 mAb or with nonspecific isotypic IgG (control) prior to being reacted with the κB probe. ns: non-specific binding.

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