Basak, et al. Supplementary Information

-1-

Cell, Volume 128

Supplemental Data

A Fourth IκκκκB Protein within the NF-κκκκB Signaling Module

Soumen Basak, Hana Kim, Jeffrey D. Kearns, Vinay Tergaonkar, Ellen O’Dea, Shannon L. Werner, Chris A. Benedict,

Carl F. Ware, Gourisankar Ghosh, Inder M. Verma, Alexander Hoffmann

Figure S1. Specificity of LTββββR agonist induced NF-κκκκB signaling in MEF A. Extensive time course analysis on agonist treated wild type MEF revealed temporal profile of NF-

κB DNA binding activities downstream of LTβR. We identified two major complexes as the

RelA (arrow) and RelB (arrowhead) dimer bound to DNA. Onset of activation for both RelA and

RelB dimer was observed by EMSA at 3-5hrs post-stimulation.

B. Specificity of NF-κB response mediated by LTβR agonist was determined by utilizing ltβr-/- MEF. DNA binding complexes were resolved on EMSA that revealed lack of activation of both the NF-

κB dimers in receptor knock out cells (lanes 5-8).

C. Composition of RelA and RelB dimer induced by 0.3µg/ml LTβR agonist was evaluated at 36hr

post-stimulation by super-shift analysis. Results indicated accumulation of RelB:p52 as major NF-

κB activity at the late phase.

D. Nuclear DNA binding activity induced by 0.3 µg/ml LTβR agonist was compared with that

induced by 1ng/ml TNF. TNF-treated nuclear extract was serially diluted with nuclear extract

derived from nfκb1-/-crel-/-rela-/-MEFs (nfkb-/-) that lack detectable κB-site DNA binding activity to maintain uniformity of total nuclear protein in different lanes.

E. Amplitude of LTβR agonist induced NF-κB response was evaluated by comparing κB-DNA

binding activity induced by 0.3µg/ml receptor agonist (lane 5) with that induced by different

concentrations of TNF (lanes 1-4).

F. Specificity of NF-κB response mediated by LTβR agonist was determined by utilizing three

different previously described LTβR antibody, AF.H6, 4H8 and an antibody cocktail of 4H8 and

3C8 (10:1). NF-κB activation scored by EMSA revealed similar activation profile and dimer

composition in wild type MEF.

G. Nuclear localization of indicated NF-κB proteins was monitored by immunoblotting with specific

antibodies in extracts prepared from TNF (1ng/ml) or LTβR agonist (0.3µg/ml) stimulated cells.

Antibodies against TAF20 and tubulin were employed as nuclear and cytoplasmic markers.

H. Immunoblots for p105/p50 or p100/p52 proteins of extracts prepared from TNF or LTβR

stimulated cells. Signals for p105 or p100 were quantitated, normalized to the actin loading

control and graphed relative to signal levels in resting cells (bottom panel).

I. Phospho-IκBα immunoblot of extracts prepared from cells treated with proteasome inhibitor, MG-

132 alone (top panel) or in combination with 0.3µg/ml LTβR antibody (bottom panel). An

antibody that recognizes phosphorylated serine-32 and serine-36 of IκBα was used.

J. Phospho-IκBα immunoblot of extracts prepared from cells treated with different concentrations of

TNF in presence of MG-132 to reveal sensitivity of this assay.

Basak, et al. Supplementary Information

-2-

A B

D

5 25

125

625

0

Fold dilutionwith nfκκκκb-/-

extracts

TNF 1ng/ml

nf κκ κκb-/-

αα αα-LTββ ββR

1 2 3 4 5 6 7 *

C

wt ltββββr-/-

15- .5 5αααα-LTββββR 15- .5 5 hr

* 1 2 3 4 5 6 7 8

0.3µg/ml αααα-LTββββR

3- .5 1 5 8 24 hr

1 2 3 4 5 6 7

αααα-LTββββR36 hr

1 2 3 4

supershiftantibody p

50

p52

p50/52

–

E

1 0.1

0.01

0.001

TNF (ng/ml)

αα αα-LTββ ββR

1 2 3 4 5 *

FAF.H6 4H8 4H8+3C8

αααα-LTββββR 0 .5 5 15 hr24 0 .5 5 15 24 0 .5 5 15 24

* 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

G

RelA

RelB

TAF20

.25 .5 5 150

TNF αααα-LTββββR

hr

tubulin1 2 3 4 5

H

p105 p100

0 1 16842.5.25 0 1 16 hr842.5.25[protein] (%

)

0

50

100

p105

p50

p100

p52

actin

TNF

1ng/ml

αααα-LTββββR

10µg/ml

αααα-LTββββR

0.3µg/ml

0 .5 5 15 0 .5 5 15 hr0.251.25

1 2 3 4 5 6 7 8 9 10 11

I

J

0.11.0

0.01

––

MG-132

TNF 10 min ng/ml

p-IκκκκBαααα1 2 3 4 5

(–)

αααα-LTββββR0.3 µµµµg/ml

0 0.5 1 3 5 hr

MG-132

p-IκκκκBαααα

1 2 3 4 5

p-IκκκκBαααα

Basak, et al. Supplementary Information

-3-

Figure S2. nfκκκκb2 p100 binds to and inhibits NF-κκκκB/RelA DNA binding activity

A. Immunoblot revealing the expression level of RelA in IκB-deficient cells (lane 3) and IκB/RelA-

deficient cells reconsitituted with a retroviral transgene expressing TAP-tagged p65 (lane 3).

B. RelA protein associated with p105 and p100 were examined by RelA immunoblotting of

immunoprecipitates from wild type (left panel) or IκB-deficient (right panel) MEF extracts using

ankyrin domain-specific antibodies recognizing p105 and p100.

C. Specificity and identity of the NF-κB DNA binding activities as revealed upon 0.8% DOC

treatment of cytosolic extract was ascertained by competition with unlabelled oligonucleotide

containing the κB site or a mutated site (lanes 2-3), and by supershift analysis using the indicated

antibodies (lanes 4-9). We identified major latent DNA binding complexes (arrows) as the

RelA:p50 dimer in both wild type (top panel) as well as IκB-deficient (bottom panel) cells.

D. NF-κB DNA binding activity induced by TNF was examined by EMSA in nuclear extracts

prepared at indicated times from HEK293T cells (lanes 1-3) or HEK293T cells previously

transfected with a p100 expression vector (lanes 4-5).

E. NF-κB DNA binding activity (arrow) was monitored by EMSA following stable transduction of

IκB-deficient cells with lentivirus expressing indicated control shRNA.

TAP-p65

p65

A

TAP-p65

iκκκκb-/-rela-/-

– –

iκκκκb-/-

1 2 3

B

p105c IP

p100c IP

p105

p100

RelA* *

lysate

lysate**

p105c IP

p100c IP

lysate

lysate

iκκκκb-/-wt

1 2 3 4 5 6 7 8

RelA

RelB

cRel

p50

p52

wt

mt

p50/52

–

Wild type

i κκ κκb-/-

antibodiesoligoC

1 2 3 4 5 6 7 8 9

D

0TNF 15 30 15 30 min

p100 –

1 2 3 4 5

1.4 4.4 2.2

control shRNA #

–

1 2 3 4

E

Basak, et al. Supplementary Information

-4-

Figure S3. nfκκκκb2 p100 is required and sufficient for LTββββR activation of NF-κκκκB/RelA. A. Degradation of p100 and accumulation of p52 in nfkb2

-/- MEF reconstituted with a p100

expressing retrovirus (pBabe-p100) was monitored by immunoblotting using an N-terminal

p100/p52 antibody during a stimulation time course with α-LTβR antibody.

B. Nuclear localization of indicated NF-κB proteins was monitored by immunoblotting extracts

prepared from TNF (1ng/ml) or LTβR agonist (0.3µg/ml) stimulated IκB-deficient cells with

specific antibodies.

C. Specificity and composition of NF-κB DNA binding complexes induced by LTβR stimulation in

iκbα-/-β-/-ε-/- cells was determined by oligonucleotide competition (lanes 2-3) and supershift with the indicated antibodies (lanes 4-7). RelA- (arrow) and RelB- (arrowhead) containing complexes

are indicated.

D. Composition of RelA and RelB DNA binding dimer present in resting (top panel) or LTβR-

induced (bottom panel) IκB-deficient (iκbα-/-β-/-ε-/-) cells was examined by supershift analysis (lanes 2-4). At 5hr of stimulation both RelA:p50 (arrow) and RelB:p50 (arrowhead) dimer was

induced.

E. Immunoblots for p105 (top panels) and p100 (bottom panels) of lysates prepared from TNF (left

panels) and LTβR (right panels) stimulated IκB-deficient cells.

F. Association of p100 and RelA during LTβR signaling was monitored by p100 immunoblotting of

RelA co-immunoprecipitates performed with extracts prepared from IκB-deficient cells at the

indicated times.

G. Probing the nature of the p100-RelA interaction in canonical IκB-deficient cells (iκbα-/-β-/-ε-/-)

before and after LTβR stimulation, using the DOC sensitivity assay as shown in Figure 4a for wild

type cells. Results indicate that p100 forms DOC sensitive complexes in IκB-deficient cells with

the RelA:p50 dimer that respond to LTβR signaling.

Basak, et al. Supplementary Information

-5-

0 .3 .8DOC 0 .3 .8 0 .3 .8 0 .3 .8 %

p50

RelA

p100

αααα-LTββββR, 5hr–

IPWash IPWash

1 2 3 4 5 6 7 8 9 10 11 12

G

150 .5 5αααα-LTββββR 15 hr0 .5 5

p100

p52

nfκκκκb2-/-nfκκκκb2-/-

p100wt

1 2 3 4 5 6 7 8

A

RelA

RelB

TAF20

.25 .5 5 150

TNF αααα-LTββββR

hr

tubulin

1 2 3 4 5

B

C

p50

p52

p50/52

–

0 hr

5 hr

1 2 3 4

TNF αααα-LTββββR

0 .5 5 15 hr0 .25 1.25

p105

p50

p100

p52

actin

1 2 3 4 5 6 7

E

RelA

p100

p50*

αααα-LTββββR

1 2 3 4

0 0.5 5 15 hr

iκκκκb-/-RelA IP

F

supershiftantibody

Dall

RelA

RelB

cRel

oligo antibody

1 2 3 4 5 6 7

– wt

mt

Basak, et al. Supplementary Information

-6-

Figure S4. Upstream signaling mediators of LTββββR responses.

A. Degradation of p100 and p52 accumulation in response to LTβR stimulation was monitored by

immunoblotting in ikk1-/- MEF (lanes 1-4) or ikk1

-/- MEF reconstituted with an IKK1-expressing

retrovirus (lanes 5-8).

B. Requirement of functional NIK protein, which is able to induce p100 processing, in LTβR

mediated NF-κB response was examined by utilizing NIK-aly MEF. DNA binding complexes

were resolved on EMSA to reveal lack of activation of both the NF-κB dimers in NIK-aly MEF.

C. Immunoblot for NIK of lysates prepared from IκB-deficient cells and such cells transduced with a

NIK shRNA expressing lentivirus revealed efficient knockdown of NIK expression. The actin

immunoblot (bottom panel) serves as a loading control.

D. Requirement of NEMO in TNF induction of NF-κB activity was examined by stimulating wild

type or nemo-/- MEFs with 1ng/ml TNF. Nuclear extracts were prepared at 30min post-stimulation

to reveal lack of TNF induced κB DNA binding activity in absence of NEMO.

E. Dimer composition of NF-κB activity induced in nemo-/- MEF by LTβR signaling was evaluated

by supershift analysis using indicated antibodies to reveal predominantly p50-containing DNA

binding activities within nucleus at 5hr post-stimulation.

NIK

actin

NIK-KD – +

iκκκκb-/-C

B

D E

1 2 3 4 5 6 7 8

NIK-aly

15- .5 5αααα-LTββββR 15- .5 5 hr

NIK-wt

30 min- 30 -TNF

WT

1 2 3 4

nemo-/-

p50

p52

p50/52

–

supershiftantibody

5 hr

0 hr

1 2 3 4

A

150 .5 5αααα-LTββββR 15 hr0 .5 5

p100

ikk1-/-ikk1-/-

IKK1wt

p52

1 2 3 4 5 6 7 8

Basak, et al. Supplementary Information

-7-

Figure S5. Schematic depiction of the four IκκκκB-containing NF-κκκκB signaling module. Schematic depiction of biochemical reactions described in the mathematical model version 3.0.

This model recapitulates TNF and LPS activation of NF-κB, as previously published (Werner et

al. 2005), as well as the mechanisms described in this paper for LTβR activation of the RelA:p50

dimer. Numbers indicate reactions (as shown in Table S3) that are determined by specific

biochemical rate constants and corresponding parameters (Table S2).

Basak, et al. Supplementary Information

-8-

II. Description of the Computational Model

Introduction

We previously constructed a computational model to describe NF-κB activation events in response to

IKK activation by TNFα (Hoffmann et al., 2002). This model comprised a singular NF-κB species, three

IκB isoforms IκBα/β/ε, and IKK. Synthesis and degradation of the IκBs and cellular localization and

interactions for all components are calculated using a system of ordinary differential equations. Recent

studies by our group expanded the model to include numerically defined IKK activity (Werner et al.,

2005) and inducible syntheses of IκBε and IκBβ (Kearns et al., 2006).

This version includes nfkb2 p100/IκBδ as a fourth IκB protein that is degraded in response to IKK1

activity to release NF-κB/RelA dimers. p100/IκBδ is transcriptionally regulated by NF-κB/RelA. The

scope of the model is shown schematically in Figure S6. Eight components, including free and bound

forms of p100/IκBδ and IKK1, were added to the model. Experimental data regarding the synthesis and

degradation of p100/IκBδ mRNA and protein that allowed for parameter fitting will be reported

elsewhere (V.Shih, J.D.K., S.B., A.H., unpublished observations). Three assumptions were made to fit

parameters with experimental observations: NF-κB-mediated p100/IκBδ transcription is delayed by 45

min akin to IκBε, and IκBβ (Kearns et al., 2006); p100/IκBδ synthesis is diminished in response to

activation of the non-canonical pathway by LTβ stimulation to account for p100 processing to p52; and

IκB-NF-κB association rates decrease in response to non-canonical activation.

This model is able to reproduce the general temporal profiles of p100, IκBα, IκBβ, and IκBε protein

levels and NF-κB/RelA nuclear activity as measured experimentally in response to transient TNF,

transient LPS, and persistent LTβR stimulations.

Simulation Regime

Simulations were performed in Matlab version 2006a (Mathworks) using the built-in ode15s solver at

default settings. Simulations comprised of two distinct and sequential phases. The first phase, Phase 1,

prior to the onset of stimulation (t = 0), was used to reach equilibrium; 2000 minutes was found to be

sufficient in all scenarios shown here. This represents the resting state prior to stimulation.

The second phase, Phase 2, occurs in positive time and begins with the application of a stimulatory input

to the system, represented by an increase in the active IKK component. This perturbs the equilibrium

attained in Phase 1 and allows for nuclear localization of NF-κB, subsequent induction of IκB and P100

syntheses, and the resulting negative feedbacks.

The results of simulations can be graphically plotted within MatLab or within a Microsoft Excel

spreadsheet. The model figures in the paper were plotted within Excel. A multidimensional array of

concentration values for each component of the model at 1 min intervals is constructed within the MatLab

model and then exported to a tab-delimited text file. This file is then imported into an Excel spreadsheet.

Basak, et al. Supplementary Information

-9-

Table S1: Model Components

Proteins and Protein Complexes Model Component Localization

IKK2 IKK Cytoplasmic

IKK1 IKK1 Cytoplasmic

NF-κB NFkB Cytoplasmic

NF-κB NFkBn Nuclear

IκBα IkBa Cytoplasmic

IκBα IkBan Nuclear

IκBβ IkBb Cytoplasmic

IκBβ IkBbn Nuclear

IκBε IkBe Cytoplasmic

IκBε IkBen Nuclear

P100 P100 Cytoplasmic

P100 P100n Nuclear

IκBα mRNA IkBat Cytoplasmic

IκBβ mRNA IkBbt Cytoplasmic

IκBε mRNA IkBet Cytoplasmic

P100 mRNA P100t Cytoplasmic

IKK2-IκBα IKKIkBa Cytoplasmic

IKK2-IκBβ IKKIkBb Cytoplasmic

IKK2-IκBε IKKIkBe Cytoplasmic

IKK1-P100 P100IKK1 Cytoplasmic

IκBα-NF-κB IkBaNFkB Cytoplasmic

IκBα-NF-κB IkBaNFkBn Nuclear

IκBβ-NF-κB IkBbNFkB Cytoplasmic

IκBβ-NF-κB IkBbNFkBn Nuclear

IκBε-NF-κB IkBeNFkB Cytoplasmic

IκBε-NF-κB IkbeNFkBn Nuclear

P100-NF-κB P100NFkB Cytoplasmic

P100-NF-κB P100NFkBn Nuclear

IKK2-IκBα-NF-κB IKKIkBaNFkB Cytoplasmic

IKK2-IκBβ-NF-κB IKKIkBbNFkB Cytoplasmic

IKK2-IκBε-NF-κB IKKIkBeNFkB Cytoplasmic

IKK1-P100-NF-κB P100IKK1NFkB Cytoplasmic

Table S1: The model contains thirty-two components with distinct nuclear and cytoplasmic localizations.

Free IKK1/2 and complexes containing IKK1/2 as well as P100/IκB mRNA are exclusively cytoplasmic.

P100, IκB and NF-κB exist in free and bound forms in both the cytoplasm and the nucleus. Free P100,

IκB and NF-κB are allowed to shuttle between nucleus and cytoplasm while shuttling of inhibitor-NF-κB

complexes is limited to nuclear export.

Basak, et al. Supplementary Information

-10-

Table S2: Model Parameters

Table S2A: Parameter Nomenclature

Each parameter begins with a one or two-letter code that defines the reaction that the parameter controls.

Code Reaction Type

A Protein-Protein Complex Association

D Protein-Protein Complex Dissociation

ex Nuclear Export

H Hill Coefficient

in Nuclear Import

pd Protein Degradation

ps Protein Synthesis

rd RNA Degradation

rsr RNA Synthesis Regulated (inducible)

rsu RNA Synthesis Unregulated (constitutive)

An optional second one-letter code is used to define cellular localization.

Code Localization

C Nuclear

N Cytoplasmic

The last code defines the model components involved in the reaction. A numerical prefix is used to

define a reaction involving a complex containing the following N components.

Code Reaction Type

a IkBa

b IkBb

e IkBe

P P100

n NFkB

i IKK (IKK2)

q IKK1

2 Complex of 2 Components

3 Complex of 3 Components

Basak, et al. Supplementary Information

-11-

Table S2B: Complex Association Rates

Parameter ka (µµµµM

-1 min

-1)

a_c_an 30

a_n_an 30

a_c_bn 30

a_n_bn 30

a_c_en 30

a_n_en 30

a_c_pn 30

a_n_pn 30

a_c_ai 1.35

a_c_bi 0.36

a_c_ei 0.54

a_c_pq 0.54

a_c_2ani 11.1

a_c_2bni 2.88

a_c_2eni 4.2

a_c_2pnq 4.2

a_c_2ain 30

a_c_2bin 30

a_c_2ein 30

a_c_2pqn 30

Table S2C: Complex Dissociation Rates

Parameter kd (min

-1)

d_c_an 6 E-05

d_n_an 6 E-05

d_c_bn 6 E-05

d_n_bn 6 E-05

d_c_en 6 E-05

d_n_en 6 E-05

d_c_pn 6 E-05

d_n_pn 6 E-05

d_c_ai 0.075

d_c_bi 0.105

d_c_ei 0.105

d_c_pq 0.105

d_c_2ani 0.075

d_c_2bni 0.105

d_c_2eni 0.105

d_c_2pnq 6 E-05

d_c_2ain 6 E-05

d_c_2bin 6 E-05

d_c_2ein 6 E-05

d_c_2pqn 6 E-05

Table S2D: Nuclear Import and Export Translocation Rates

Parameter Rate (min

-1)

in_a 0.09

ex_a 0.012

in_b 0.009

ex_b 0.012

in_e 0.045

Basak, et al. Supplementary Information

-12-

ex_e 0.012

in_p 0.045

ex_p 0.012

in_n 5.4

ex_n 0.0048

in_2an 0.276

in_2bn 0.0276

in_2en 0.138

in_2pn 0.276

ex_2an 0.828

ex_2bn 0.414

ex_2en 0.414

ex_2pn 0.414

Table S2E: Protein Degradation Rates

Parameter kdeg (min

-1)

pd_c_a 0.12

pd_n_a 0.12

pd_c_2an 6 E-05

pd_n_2an 6 E-05

pd_c_3ain 0.36

pd_c_2ai 0.0018

pd_c_b 0.18

pd_n_b 0.18

pd_c_2bn 6 E-05

pd_n_2bn 6 E-05

pd_c_3bin 0.12

pd_c_2bi 6 E-04

pd_c_e 0.18

pd_n_e 0.18

pd_c_2en 6 E-05

pd_n_2en 6 E-05

pd_c_3ein 0.18

pd_c_2ei 0.0018

pd_c_p 0.12

pd_n_p 0.18

pd_c_2pn 6 E-05

pd_n_2pn 6 E-05

pd_c_3pqn 0.18

pd_c_2pq 0.0012

Table S2F: Transcription Rates

Parameter Rate (min

-1)

rsu_a 1.1848 E-04

rsu_b 4.272 E-05

rsu_e 6.1 E-06

rsu_p 6.1 E-06

Basak, et al. Supplementary Information

-13-

Parameter Rate (µµµµM-1 min

-1)

rsr_an 9.504

rsr_bn 0.02

rsr_en 1.28

rsr_pn 1.6

Parameter Hill Coefficient for NFkB inducible transcription Value

h_an IkBa 3.0

h_bn IkBb 3.0

h_en IkBe 3.0

h_pn P100 3.0

Parameter Delay for NFkB inducible transcription min

rsr_delay_a IkBa 0.0

rsr_delay_b IkBb 45.0

rsr_delay_e IkBe 45.0

rsr_delay_p P100 90.0

Table S2G: Transcript Degradation

Parameter kd (min

-1)

rd_a 0.0252

rd_b 0.00336

rd_e 0.00672

rd_p 0.00168

Table S2H: Translation Rates

Parameter Rate (min

-1)

ps_c_a 0.02448

ps_c_b 0.02448

ps_c_e 0.02448

ps_c_p 0.02448

Basak, et al. Supplementary Information

-14-

Table S3: Summary of Reactions

Reaction Associated Parameter(s) Category Location

1 IkBa + IKK => IKKIkBa a_c_ai Association Cytoplasm

2 IkBa + NFkB => IkBaNFkB a_c_an Association Cytoplasm

3 IkBb + IKK => IKKIkBb a_c_bi Association Cytoplasm

4 IkBb + NFkB => IkBbNFkB a_c_bn Association Cytoplasm

5 IkBe + IKK => IKKIkBe a_c_ei Association Cytoplasm

6 IkBe + NFkB => IkBeNFkB a_c_en Association Cytoplasm

7 P100 + IKK1 => P100IKK1 a_c_pq Association Cytoplasm

8 P100 + NFkB => P100NFkB a_c_pn Association Cytoplasm

9 IKKIkBa + NFkB => IKKIkBaNFkB a_c_2ain Association Cytoplasm

10 IkBaNFkB + IKK => IKKIkBaNFkB a_c_2ani Association Cytoplasm

11 IKKIkBb + NFkB => IKKIkBbNFkB a_c_2bin Association Cytoplasm

12 IkBbNFkB + IKK => IKKIkBbNFkB a_c_2bni Association Cytoplasm

13 IKKIkBe + NFkB => IKKIkBeNFkB a_c_2ein Association Cytoplasm

14 IkBeNFkB + IKK => IKKIkBeNFkB a_c_2eni Association Cytoplasm

15 P100IKK1 + NFkB => P100IKK1NFkB a_c_2pqn Association Cytoplasm

16 P100NFkB + IKK1 => P100IKK1NFkB a_c_2pnq Association Cytoplasm

17 IkBan + NFkBn => IkBaNFkBn a_n_an Association Nucleus

18 IkBbn + NFkBn => IkBbNFkBn a_n_bn Association Nucleus

19 IkBen + NFkBn => IkBeNFkBn a_n_en Association Nucleus

20 P100n + NFkBn => P100NFkBn a_n_pn Association Nucleus

21 IKKIkBa => IkBa + IKK d_c_ai Dissociation Cytoplasm

22 IkBaNFkB => IkBa + NFkB d_c_an Dissociation Cytoplasm

23 IKKIkBb => IkBb + IKK d_c_bi Dissociation Cytoplasm

24 IkBbNFkB => IkBb + NFkB d_c_bn Dissociation Cytoplasm

25 IKKIkBe => IkBe + IKK d_c_ei Dissociation Cytoplasm

26 IkBeNFkB => IkBe + NFkB d_c_en Dissociation Cytoplasm

27 P100IKK1 => P100 + IKK d_c_pq Dissociation Cytoplasm

28 P100NFkB => P100 + NFkB d_c_pn Dissociation Cytoplasm

29 IKKIkBaNFkB => IKKIkBa + NFkB d_c_2ain Dissociation Cytoplasm

30 IKKIkBaNFkB => IkBaNFkB + IKK d_c_2ani Dissociation Cytoplasm

31 IKKIkBbNFkB => IKKIkBb + NFkB d_c_2bin Dissociation Cytoplasm

32 IKKIkBbNFkB => IkBbNFkB + IKK d_c_2bni Dissociation Cytoplasm

33 IKKIkBeNFkB => IKKIkBe + NFkB d_c_2ein Dissociation Cytoplasm

34 IKKIkBeNFkB => IkBeNFkB + IKK d_c_2eni Dissociation Cytoplasm

35 P100IKK1NFkB => P100IKK1 + NFkB d_c_2pqn Dissociation Cytoplasm

36 P100IKK1NFkB => P100NFkB + IKK1 d_c_2pnq Dissociation Cytoplasm

37 IkBaNFkBn => IkBan + NFkBn d_n_an Dissociation Nucleus

38 IkBbNFkBn => IkBbn + NFkBn d_n_bn Dissociation Nucleus

39 IkBeNFkBn => IkBen + NFkBn d_n_en Dissociation Nucleus

40 P100NFkBn => P100n + NFkBn d_n_pn Dissociation Nucleus

41 IkBan => IkBa ex_a Export -

42 IkBbn => IkBb ex_b Export -

43 IkBen => IkBe ex_e Export -

44 P100n => P100e ex_p Export -

45 NFkBn => NFkB ex_n Export -

46 IkBaNFkBn => IkBaNFkB ex_2an Export -

47 IkBbNFkBn => IkBbNFkB ex_2bn Export -

48 IkBeNFkBn => IkBeNFkB ex_2en Export -

49 P100NFkBn => P100NFkB ex_2pn Export -

50 IkBa => IkBan in_a Import -

51 IkBb => IkBbn in_b Import -

52 IkBe => IkBen in_e Import -

53 P100 => P100n in_p Import -

Reaction Associated Parameter(s) Category Location

Basak, et al. Supplementary Information

-15-

54 NFkB => NFkBn in_n Import -

55 IkBaNFkB => IkBaNFkBn in_2an Import -

56 IkBbNFkB => IkBbNFkBn in_2bn Import -

57 IkBeNFkB => IkBeNFkBn in_2en Import -

58 P100NFkB => P100NFkBn in_2pn Import -

59 IkBa => pd_c_a Prot. deg. Cytoplasm

60 IkBb => pd_c_b Prot. deg. Cytoplasm

61 IkBe => pd_c_e Prot. deg. Cytoplasm

62 P100 => pd_c_p Prot. deg. Cytoplasm

63 IKKIkBa => IKK pd_c_2ai Prot. deg. Cytoplasm

64 IkBaNFkB => NFkB pd_c_2an Prot. deg. Cytoplasm

65 IKKIkBb => IKK pd_c_2bi Prot. deg. Cytoplasm

66 IkBbNFkB => NFkB pd_c_2bn Prot. deg. Cytoplasm

67 IKKIkBe => IKK pd_c_2ei Prot. deg. Cytoplasm

68 IkBeNFkB => NFkB pd_c_2en Prot. deg. Cytoplasm

69 P100IKK1 => IKK1 pd_c_2pq Prot. deg. Cytoplasm

70 P100NFkB => NFkB pd_c_2pn Prot. deg. Cytoplasm

71 IKKIkBaNFkB => IKK + NFkB pd_c_3ain Prot. deg. Cytoplasm

72 IKKIkBbNFkB => IKK + NFkB pd_c_3bin Prot. deg. Cytoplasm

73 IKKIkBeNFkB => IKK + NFkB pd_c_3ein Prot. deg. Cytoplasm

74 P100IK1NFkB => IKK1 + NFkB pd_c_3pqn Prot. deg. Cytoplasm

75 IkBaNFkBn => NFkBn pd_n_2an Prot. deg. Nucleus

76 IkBbNFkBn => NFkBn pd_n_2bn Prot. deg. Nucleus

77 IkBeNFkBn => NFkBn pd_n_2en Prot. deg. Nucleus

78 P100NFkBn => NFkBn pd_n_2pn Prot. deg. Nucleus

79 IkBan => pd_n_a Prot. deg. Nucleus

80 IkBbn => pd_n_b Prot. deg. Nucleus

81 IkBen => pd_n_e Prot. deg. Nucleus

82 P100n => pd_n_p Prot. deg. Nucleus

83 => IkBa ps_c_a Prot. synth. Cytoplasm

84 => IkBb ps_c_b Prot. synth. Cytoplasm

85 => IkBe ps_c_e Prot. synth. Cytoplasm

86 => P100 ps_c_p Prot. synth. Cytoplasm

87 IkBat => rd_a RNA deg. -

88 IkBbt => rd_b RNA deg. -

89 IkBet => rd_e RNA deg. -

90 P100t => rd_p RNA deg. -

91 => IkBat (induced by NF-IB) rsr_an, rsr_delay_a, h_an RNA synth. -

92 => IkBbt (induced by NF-IB) rsr_bn, rsr_delay_b, h_bn RNA synth. -

93 => IkBet (induced by NF-IB) rsr_en, rsr_delay_e, h_en RNA synth. -

94 => P100t (induced by NF-IB) rsr_pn, rsr_delay_p, h_pn RNA synth. -

95 => IkBat (constitutive) rs_a RNA synth. -

96 => IkBbt (constitutive) rs_b RNA synth. -

97 => IkBet (constitutive) rs_e RNA synth. -

98 => P100t (constitutive) rs_p RNA synth. -

Inducible syntheses of IkBb (#92), IkBe (#93), and P100 (#94) utilize previous values of nuclear NF-kB

to affect a transcriptional delay in Phase 2. The logic is as follows:

If Time <= Delay, Inducible Transcription = Inducible parameter * NFkB(Time=0)

If Time > Delay, Inducible Transcription = Inducible parameter * NFkB(Time - Delay)

Basak, et al. Supplementary Information

-16-

Simulation Conditions

Table S4: Initial System Conditions

Component Localization Concentration (µµµµM)

NFkB Cytoplasmic 0.0

NFkBn Nuclear 0.0

IkBa Cytoplasmic 0.0

IkBan Nuclear 0.0

IkBb Cytoplasmic 0.0

IkBbn Nuclear 0.0

IkBe Cytoplasmic 0.0

IkBen Nuclear 0.0

P100 Cytoplasmic 0.0

P100n Nuclear 0.0

IkBat Cytoplasmic 0.0

IkBbt Cytoplasmic 0.0

IkBet Cytoplasmic 0.0

P100t Cytoplasmic 0.0

IKKIkBa Cytoplasmic 0.0

IKKIkBb Cytoplasmic 0.0

IKKIkBe Cytoplasmic 0.0

P100IKK1 Cytoplasmic 0.0

IkBaNFkB Cytoplasmic 0.075

IkBaNFkBn Nuclear 0.0

IkBbNFkB Cytoplasmic 0.01875

IkBbNFkBn Nuclear 0.0

IkBeNFkB Cytoplasmic 0.025

IkbeNFkBn Nuclear 0.0

P100NFkB Cytoplasmic 0.00625

P100NFkBn Nuclear 0.0

IKKIkBaNFkB Cytoplasmic 0.0

IKKIkBbNFkB Cytoplasmic 0.0

IKKIkBeNFkB Cytoplasmic 0.0

P100IKK1NFkB Cytoplasmic 0.0

IKK Cytoplasmic 0.1

IKK1 Cytoplasmic 0.1

References

Hoffmann, A., A. Levchenko, M.L. Scott, and D. Baltimore. 2002. The IkappaB-NF-kappaB signaling

module: temporal control and selective gene activation. Science. 298:1241-5.

Kearns, J., S. Basak, S. Werner, C. Huang, and A. Hoffmann. 2006. IkBe provides negative feedback to

control NF-kB oscillations, signaling dynamics, and inflammatory gene expression. Journal of

Cell Biology. in press.

Werner, S., D. Barken, and A. Hoffmann. 2005. Stimulus specificity of gene expression programs

determined by temporal control of IKK activity. Science. 309:1857-61.