RelB RHR stabilization by p52 in vitro. A, Western
blot analysis of the steady state levels of RelB RHR and RelB DD proteins in
HEK 293 cells transfected with FLAG-p100, FLAG-p52, and RelB-GFP in different
combinations. In the presence of p100 and p52, RelB protein levels are
enhanced. B, Coomassie-stained SDS-PAGE showing purity of p52-RelB
heterodimer, p52 homodimer, and RelB RHR homodimer. A lower concentration of
p52-RelB heterodimer is shown in lane 4 to visualize the separation
between the two proteins. RelB continuously degrades during purification, and
the degradation products are seen in the gel (lane 4). Aggregated
species of RelB in lane 3 is marked by an asterisk.
C, co-refolded mixtures of RelB RHR and p52 RHR (black), and
RelB RHR and p52 DD (gray) were separated by cation exchange
(S-Sepharose column) chromatography. D, Coomassie-stained SDS-PAGE of
samples from the S column chromatography of the RelB RHR-p52 DD (top
panel) and RelB RHR-p52 RHR (bottom panel). The load appears to
contain excess p52, which masks the RelB RHR. E, Western blot
analysis of the steady state levels of p100/p52 (top panel) and RelB
(middle panel) in wt, nfkb2-/- MEF and
p52-reconstituted nfkb2-/- cells. Reconstituted p52
migrates higher due to the exact site of processing of p100 is unknown.
F, DNA binding-defective p52 mutant stabilizes RelB. Western blot
analysis of the steady state levels of p100/p52 (top panel) and RelB
(middle panel) in wt, nfkb2-/- MEF, and
p52-reconstituted nfkb2-/- cells. Reconstituted p52
migrates higher due to the exact site of processing of p100 is unknown.
G, electrophoretic mobility shift assay analysis of wild type p52 and
p52 R54A,Y55A double mutant. Lanes 2 and 6, 3 and
7, 4 and 8, and 5 and 9 contain
1100, 250, 25, and 2.5 nm wild type and double mutant p52,
respectively.