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. Author manuscript; available in PMC: 2010 Jan 15.
Published in final edited form as: Biochem J. 2009 Jan 15;417(2):457–466. doi: 10.1042/BJ20080925

Figure 4. BCL11B enhances nuclear levels of p50 and p65, as well as binding of NF-kB consensus sites following T cell activation.

Figure 4

(A) EMSA using a labeled consensus NF-kB oligonucleotides and nuclear extracts prepared from Jurkat cells stimulated (lanes 2 to 8) or not (lane 1) with 50 ng/ml PMA and 1 μM Ionomycin for 1 hr. For the supershift experiments the nuclear extracts were incubated with the indicated antibodies for 15 min prior to incubation with the labeled oligonucleotide probe. (B) MSCV and MSCV-BCL11B Jurkat cells were stimulated or not with 50 ng/ml PMA and 1 μM Ionomycin for 1 hr (left panel), or with anti-CD3 and anti-CD28 (right panel). The nuclear extracts were analyzed by immunoblotting for p65 and p50 proteins. Actin was used as a loading control. Numbers indicate fold increase in the levels of p50 and p65 after normalization to actin, evaluated through densitometry. (C) EMSA using labeled consensus NF-kB oligonucleotides and nuclear extracts prepared from MSCV (lanes 1 through 4) and MSCV-BCL11B (lanes 5 through 8) Jurkat cells stimulated (lanes 2 to 4 and 6 to 8) or not (lanes 1 and 5) with 50 ng/ml PMA and 1 μM Ionomycin for 1 hr. For the supershift experiments the nuclear extracts were incubated with the indicated antibodies for 15 min prior to incubation with the labeled probe. NFkB-DNA complexes are indicated by the black arrow and the supershifted complexes by the gray arrow. Enhancement of binding as a result of BCL11B overexpression is indicated by asterisks.