Supplementary Figure legends
Supplementary Figure 1.
HeLa/vector, HeLa/RelA-WT, or HeLa/RelA-SA cells were transfected with scramble
siRNA or RelA(5’UTR) siRNA. Cell lysates were immunoblotted with anti-Flag or
anti-tubulin. Total RNA samples were analyzed by RT-PCR using endogenous
RelA/p65-specific or GAPDH-specific primers.
Supplementary Figure 2.
HeLa/vector, HeLa/RelA-WT, or HeLa/RelA-SA cells were treated with 20 ng/ml
TNF for indicated times. Cell lysates were subjected to immunoprecipitation with
anti-Flag followed by immunoblot analysis with anti-phospho-RelA(Ser276) or
anti-Flag. Lysates were also analyzed by immunoblotting with anti-tubulin.
Supplementary Figure 3.
U2OS cells were transfected with scramble siRNA or Pim-1 siRNA. After 48 h, cells
were treated with 20 ng/ml TNF for indicated times. Cell lysates were subjected to
immunoblot analysis with anti-phospho-RelA(Ser276), anti-RelA/p65, anti-IB,
anti-Pim-1, or anti-tubulin. A ratio of RelA/p65 expression was determined by the
densitometric analysis using ‘Image J’ program. The gene expression in control cells
transfected with scramble siRNA was defined as 1.00. Total RNA samples were
analyzed by RT-PCR using RelA/p65-specific, IB-specific, Pim-1-specific,
c-IAP1-specific, or GAPDH-specific primers.
Supplementary Figure 4.
HeLa cells were transfected with scramble siRNA or Pim-1 siRNA. After 48 h, cells
were treated with 10 ng/ml Interleukin-1 for indicated times. Cell lysates were
subjected to immunoblot analysis with anti-phospho-RelA(Ser276), anti-RelA/p65,
anti-IB, anti-Pim-1, or anti-tubulin.
Supplementary Figure 5.
HeLa cells were transfected with scramble siRNA or Pim-2 siRNA. After 48 h, cells
were treated with 20 ng/ml TNF for 1 h. Cell lysates were subjected to immunoblot
analysis with anti-phospho-RelA(Ser276), anti-RelA/p65, anti-IB, anti-Pim-2, or
anti-tubulin.
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Supplementary Figure 6.
HeLa cells were left untreated or treated with TNF. Cell lysates were
immunoprecipitated with IgG or anti-Pim-1. Immunoprecipitates were subjected with
immunoblot analysis with anti-RelA/p65 or anti-Pim-1. IgL indicates immunoglobulin
light chain. Lysates were also immunoblotted with anti-RelA/p65, anti-Pim-1, or
anti-tubulin.
Supplementary Figure 7.
U2OS cells were transfected with scramble siRNA or Pim-1 siRNA. Cells were treated
with 20 ng/ml TNF for indicated times. Soluble chromatin was subjected to ChIP
analysis using anti-RelA/p65 or IgG for IB promoter binding.
Supplementary Figure 8.
293 cells expressing pNF-B-luc were transfected with scramble siRNA or PKAc
siRNA. Cells were then treated with TNF for 8 h. Luciferase assays were performed
and the results are represented as mean ± S.D. obtained from three independent
experiments, each performed in triplicate. n.s. indicates ‘not significant’. Cell lysates
were subjected to immunoblot analysis with anti-PKAc or anti-tubulin.
Supplementary Figure 9.
U2OS cells were transfected with scramble siRNA or Pim-1 siRNA. After 48 h, cells
were left untreated or treated with 100 ng/ml TNF for 24 h. Apoptotic cells were
quantitated by TUNEL assays. The results are represented as the percentage of
TUNEL-positive cells. The results are represented as the percentage of TUNEL-positive
cells. The data indicate the mean ± S.D. obtained from two independent experiments
each performed in triplicate. One asterisk indicates p < 0.05.
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