In the presence of a p50 antibody, the DNA-binding activities of NF-κB showed a super shift.
Furthermore, addition of Ro60 and La together resulted in a super-shift of the hY1 RNA band above the position obtained with either protein alone (Fig. 5C, compare lane 12 with lanes 4 and 8).
The bound protein was identified as RUNX1 by specific antibody (Fig. 2b, lane 5) in a super-shift assay and the specificity was confirmed by competitive inhibition with unlabelled wt-probe (Fig. 2c, lane 2 5) or SNP1 (variant 1) probe (Fig. 2c, lane 6 9).
The addition of anti-HIF-1α mAb resulted in an obvious super shift indicative of the union of HIF-1α to the DNA consensus region, suggesting the high DNA binding activity of HIF-1α transcription factor in TAT.
The addition of a YB-1 (1 µg) antibody caused a super-shift in the binding product, confirming that binding was due to this transcription factor (Figure 3F, lane 4).
We also confirmed that C1/C2 complexes indeed associate with the Sp1 transcription factor using a super-shift assay in the presence of an anti-Sp1 or anti-IgG antibodies in EMSA assays.
We performed a super-shift assay.
An unrelated antibody (against Ac H3K9) did not produce a super-shift.
To verify that the binding activity was due to the presence of Pax-5 we performed a super-shift experiment using a Pax-5 specific antibody, a control antibody or no antibody in the binding reaction.
Preincubation of the nuclear extract with p65 NF-κB antibody resulted in the super shift of the band.
To further corroborate the band shift observations, gel-supershift analysis was performed with a polyclonal antibody of CTCF, and a super-shifted band was detected (lane 5 in Fig. 4A).
