p38-MK2 signaling axis regulates RNA metabolism

after UV light-induced DNA damage

Borisova et al.

Supplementary Figure 1

a

d

−6 −4 −2 0 2 4 6

0.0

1.0

2.0

3.0

log2(UV/control)

−log

10(F

DR

)

−6 −4 −2 0 2 4 6

−6−4

−20

24

6

log2(UV/control 1)

log 2(U

V/co

ntro

l 2)

r 0.82n 10448

−6 −4 −2 0 2 4 6

−6−4

−20

24

6

log2(UV + p38i/control 1)

log 2(U

V +

p38i

/con

trol

2)

r 0.81n 10437

−6 −4 −2 0 2 4 6

0.0

1.0

2.0

3.0

log2(UV + p38i/UV)

−log

10(F

DR

)

e

f g h

40

60

80

100

0 10 20 30 40

Cel

l via

biili

ty (%

)

UV (J/m2)

Untreated

p38 inhibition

***

c

b

−6−4

−20

24

6

log 2(p

38si

RN

A+U

V/U

V) 3e−58

p38i

-inse

nsiti

ve p

hosh

osite

s

p38i

-sen

sitiv

eph

osph

osite

s

UVATMiATRiDNA-PKcsip38i

- + + + + +- - + - - -- - - + - -- - - - + -- - - - - +

01234

Nor

mal

ized

pM

K2 le

vels

pMK2 (T334)

pp38 (T180/Y182)

p38

Vinculin

UV (J/m2,1h recovery)- 10 20 40 80

53 -

41 -

41 -

130 -

pChk2 (T68)

pChk1 (S345)

Vinculin

pMK2 (T334)

UV (40 J/m2,1h recovery)

ATMiATRiDNA-PKcsip38i

- + + + + +- - + - - -- - - + - -- - - - + -- - - - - +

53 -

70 -

41 -

130 -

Supplementary Figure 1: Proteome-wide identification of p38-dependent phosphorylation

sites

a. U2OS cells were treated with increasing doses of UV light (10 - 80 J/m2) and left to recover

for 1 hour. Total cell lysates were resolved on SDS-PAGE and activation of p38 was monitored

with phospho-specific antibodies.

b. U2OS cells were pretreated for 1 hour with ATM inhibitor (KU-55933, 10 µM), ATR inhibitor

(VE-821, 1 µM), DNA-PKcs inhibitor (KU-57788, 10 µM) or p38 inhibitor (SB 203580, 10

µM) and then irradiated with UV light (40 J/m2, 1 hour recovery). Total cell lysates were

resolved on SDS-PAGE and blotted with the indicated antibodies (left). The bar plot shows

the mean and standard deviation of normalized pMK2 levels quantified from three replicate

experiments (right).

c. Cell viability was measured for mock-treated U2OS cells and cells irradiated with different

doses of UV light without and with 1 hour pretreatment with the p38 inhibitor. The plot shows

the mean and standard deviation of the results obtained in three biological replicate

experiments, each performed in three technical replicates. Two-sided Student’s t-test was used

to assess the significance (*** p value < 0.001).

d. Scatter plot shows the logarithmized SILAC ratios UV/control of quantified phosphorylation

sites in replicate experiments. The color-coding indicates the density. The Spearman’s rank

correlation was calculated to determine the experimental reproducibility.

e. Scatter plot shows the logarithmized SILAC ratios UV + p38i/control of quantified

phosphorylation sites in replicate experiments.

f. Identification of significantly regulated phosphorylation sites after p38 inhibition from two

replicate experiments using the limma algorithm. P value < 0.01 was used as cut-off to

determine phosphorylation sites that significantly increase after UV light.

g. Identification of significantly regulated phosphorylation sites after p38 inhibition from two

replicate experiments was done using the limma algorithm. P value < 0.01 was used as cut-off

to determine downregulated phosphorylation sites after p38 inhibition.

h. The box plot shows the SILAC ratio of p38i-insensitive sites and p38i-sensitive sites quantified

after transient knockdown of p38. Box plot represents the 25th to 75th quartiles with the

horizontal line representing the median value.

Supplementary Figure 2a b

−6 −4 −2 0 2 4 6

0.0

1.0

2.0

3.0

log2(UV + MK2/3/5i/UV)

−log

10(F

DR

) LightArg0/Lys0

MediumArg6/Lys4

HeavyArg10/Lys8

UV

Inhibitor

SILAC

+ +

p38DMSO DMSO

Transfection Emptyvector

Flag-Strep14-3-3

Flag-Strep14-3-3

+

Supplementary Figure 2: Phosphorylation by p38-MK2 induces 14-3-3 binding to cellular

proteins

a. Identification of significantly regulated phosphorylation sites after MK2/3/5 inhibition from

two replicate experiments was done using the limma algorithm. P value < 0.05 was used as

cut-off to determine downregulated phosphorylation sites after p38 or MK2/3/5 inhibition.

b. Schematic representation of the strategy used to identify UV light-induced, p38-dependent 14-

3-3 interaction partners. SILAC-labeled U2OS cells transfected with Flag-Strep-14-3-3 were

mock-treated, irradiated with UV light (40 J/m2, 1 hour recovery) or pretreated with the p38

inhibitor and irradiated with UV light. 14-3-3 and its binding partners were enriched using

StrepTactin sepharose, digested in-gel into peptides and peptide samples were analyzed by LC-

MS/MS.

Supplementary Figure 3a

b

PHC2

METTL18

SLC4A1AP

TROVE2

HP1BP3

CCDC82

LSM14A

AASDHPPT

RALYRBM6

HNRNPLL

DDX47CPSF1

ENAH

ACIN1NKAP

TCEAL3

TSEN34

MON1A

PRKRIR

TNKS1BP1

CHAMP1

ZNF281

MATR3 (2)

THRAP3

NASPARL2BP

RNF181SCAF11

FOXK2

ATE1

LMNA

METTL3

NPM3

TJP1

ENSA

TDP1

LUC7L3

POLD1

PRKDC

PMS2HNRNPF

RFC1

DHX9

MLH1RAD1

FANCI

EIF4EBP1CHEK1

TOPBP1

DBF4

PPM1G

CREBBP

BRD8

SNIP1

HMGA1

SRRM2

EP400

GINS2

KIF2C

SMC1A

NBNNUP107

SMC3

NCAPD2

BUB1

MCM6

UTP14A

HEATR1

UBE4BNIFK

NSFL1C TUBA1B

MAGED2TUBA1C

TBCB

RSF1

BPTFTUBA4A

NSMCE4A

RNF20

PSMD4RPP38

POP1

POP4

UBQLN1

Number of upregulated sites

1 2

Proteins with UV-upregulated sites

GO molecular function

GO cellular component

GO biological process

−3 −2 −1 0 1 2 3 4

510

1520

2530

log2(enrichment)

−log

10(q

)

nucleoplasm

poly(A) RNA binding

cytoplasm gene expressionintegral component

of membranemitotic cell cycle

nucleus cellular component disassembly involved in execution phase of apoptosiscytosolmembrane DNA damage checkpointDNA repair

programmed cell deathextracellularregion

chromatin bindingapoptotic processmRNA splicing, via spliceosome

nucleotide binding

Supplementary Figure 3: Analysis of UV light-induced phosphorylation sites

a. GO terms significantly enriched among proteins with UV light-induced phosphorylation sites.

The dot plot shows significantly overrepresented GO terms associated with proteins containing

UV light-induced phosphorylation sites compared to proteins containing non-regulated sites.

The significance of the enrichment of a specific term was determined using Fisher’s exact test.

P values were corrected for multiple hypotheses testing using the Benjamini and Hochberg

FDR.

b. Analysis of functional associations among proteins with UV light-induced S/TQ

phosphorylation sites. Functional interactions were obtained from the STRING database and

visualized using Cytoscape. Proteins with S/TQ sites not involved in functional interactions

are indicated on the right.

 

Supplementary Figure 4a

b

UV (40 J/m2, 1h recovery)

p38i - - +

Input

- + +

HEK293T RPE-1

Ponceau (14-3-3)

NELFE

pMK2 (T334)

pp38 (T180/Y182)

NELFE

Vinculin

Input

- - +

- + +

14-3-3 pull down 14-3-3 pull down

HaCaT

Input

- - + - + +

14-3-3 pull down

41 -

53 -

41 -41 -

53 -

130 -

Input

GST-14-3-3 pull down

NELFE

Ponceau (14-3-3)

NELFE

pMK2 (T334)

Vinculin

- UV H2O2

41 -

53 -

41 -

130 -

41 -

Supplementary Figure 4: MK2-dependent phosphorylation of NELFE promotes its binding

to 14-3-3

a. Validation of interaction between NELFE and 14-3-3 in HaCaT, HEK293T and RPE-1 cells.

Total cell extracts from differentially treated cells were incubated with the recombinant GST-

14-3-3. Enriched proteins were resolved by SDS-PAGE and subjected to western blotting.

b. NELFE interacts with 14-3-3 after oxidative stress induced by treatment with H2O2 for 1 hour.

Total cell extracts from differentially treated U2OS cells were incubated with the recombinant

GST-14-3-3. Enriched proteins were resolved by SDS-PAGE and subjected to western

blotting.

Supplementary Figure 5a

αDαC

αC αDR19

E92

Y85

M88

E92

R19

M88

Y85αB αA

αB

αA

C

N

N

C

E

F

HG

AB

D C I

E

F

H G

A B

DCI R19

R19E92

E92

- S I

y₁₂*

b₂Sphy₁₁

b₃A

y₁₀

b₄*D

y₉

b₅D

y₈

b₆*D

y₇

b₇L

y₆

b₈Q

y₅

b₉*E

y₄

b₁₀*S

y₃

b₁₁*S

y₂

b₁₂*R

y₁

-

m/z

y₁-NH₃158.0924

a₂173.1285

y₁175.119

b₂-H₂O183.1128

b₂201.1234

y₂-NH₃245.1244

y₂262.151

b₃*270.1448

b₄-H₂O323.1714

b₄*341.1819

y₃349.183

b₃368.1217

b₅-H₂O438.1983

b₅*456.2089

y₄-H₂O460.215

y₄-NH₃461.1991

y₄478.2256

y₉²⁺532.7282

b₆-H₂O553.2253

b₅554.1858

b₆*571.2358

y₅-H₂O588.2736

y₅-NH₃589.2576

y₅606.2842

b₇-H₂O668.2522

b₇*686.2628

y₆-H₂O701.3577

y₆-NH₃702.3417

y₆719.3682

b₈-H₂O781.3363

b₇784.2397

b₈*799.3468

y₇-H₂O816.3846

y₇-NH₃817.3686

y₇834.3952

b₈897.3237

b₉-H₂O909.3949

b₉*927.4054

y₈-H₂O931.4116

y₈-NH₃932.3956

y₈949.4221

b₁₀-H₂O1038.437

b₁₀-NH₃1039.421

y₉-H₂O1046.439

y₉-NH₃1047.423

b₁₀*1056.448

y₉1064.449

y₁₀-H₂O1117.476

b₁₁-H₂O1125.469

y₁₀1135.486

b₁₁*1143.48

y₁₁-H₂O1186.497

y₁₁-NH₃1187.481

y₁₁*1204.508

b₁₂*1230.512

y₁₂-H₂O1299.581

y₁₁1302.485

y₁₂*1317.592

050

100

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500

Rel

ativ

e in

tens

ity (%

)

NELFE pS115m/z 751.8

H. sapiens 100 EKGPVPTFQPFQR---SISADDDLQ-ESSRRPQRKSLYESF 136 M. mulatta 100 EKGPVPTFQPFQR---SISADDDLQ-ESSRRPQRKSLYESF 136P. troglodytes 100 EKGPVPTFQPFQR---SISADDDLQ-ESSRHPQRKSLYESF 136F. catus 100 EKGPVPTFQPFQR---SISADDDLQ-ESSRRPQRKSLYESF 136B. taurus 100 EKGPVPTFQPFQR---SVSADDDLQ-ESSRRPQRKSLYESF 136R. norvegicus 118 EKGPVPTFQPFQR---SMSADEDLQ-EPSRRPQRKSLYESF 154M. musculus 100 EKGPVPTFQPFQR---SMSADEDLQ-EPSRRPQRKSLYESF 136M. domestica 100 EKGPAPTFQPFQR---SISADDDLQ-ESSRRPQRKSLYESF 136T. rubripes 100 EKGPVPAFLPFQR---SVSADDE-P-ESAKRVHRKSLYESF 135D. rerio 100 EKGPAPAFLPFQR---SVSTDEE-PpDSAKRIHRKSLYESF 136X. tropicalis 100 DKGPVPSFQPFQR---SVSVDEE-QaESSRRSQRKSLYESF 136D. melanogaster 96 SETTVASYQPFsstQNDVAQETIISeIIKEEPRRQNLYQHF 136

NELFE (95-140 aa)

c

Phosphorylation site

Occ

upan

cy (%

)

0

20

40

60

80

100 ControlMK2 in vitro assay

S49 S51 S115 S251

b

d e

Supplementary Figure 5: NELFE phosphorylation by MK2 promotes its binding to 14-3-3

a. Mass spectrometric fragment ion scan of the peptide corresponding to phosphorylated serine

115 in NELFE.

b. Purified MK2 can phosphorylate immunoprecipitated NELFE on S51, S115 and S251 in vitro.

c. Conservation of the NELFE peptide sequence corresponding to serine 115 across evolution.

d. Structure of the 14-3-3 epsilon homo dimer in cartoon representation (Yellow and Cyan).

e. Topology diagrams of the 14-3-3 epsilon. Topology diagrams were prepared with TopDraw.

 

Supplementary Figure 6a b

Ratio M/L 2

Ratio H/L 2

Ratio H/M 2

Ratio M/L 3

Ratio H/L 3

Ratio H/M 3

0.71

0.64

0.6

0.59

0.54

0.51

0.68

0.62

0.58

Rat

io M

/L 1

Rat

io H

/L 1

Rat

io H

/M 1

Rat

io M

/L 2

Rat

io H

/L 2

Rat

io H

/M 2

LightArg0/Lys0

MediumArg6/Lys4

HeavyArg10/Lys8

UV

Inhibitor

SILAC

+ +

p38DMSODMSO

Chromatin proteome analysis

c ed

0

0.5

1

Unt

reat

ed UV

p38i

+ U

V

NELFE

MCM7

Ponceau

Chromatin (HaCaT)

UV (40 J/m2,1h recovery)

p38i

- + +

- - +

41 -

70 -

NELFE

MCM7

Ponceau

UV (20 J/m², recovery (h))0 1 8 24 48 72

Total cell lysate

41 -

70 -NELFE

MCM7

Ponceau

UV (40 J/m2,1h recovery)

p38i

- + +

- - +

Total cell lysate

41 -

70 -

Nor

mal

ized

NEL

FE le

vels

Supplementary Figure 6: Protein dynamics on chromatin after UV light

a. Schematic representation of the strategy used to identify UV light-induced, p38-dependent

change in the chromatin proteome. SILAC-labeled U2OS cells were mock-treated, irradiated

with UV light (40 J/m2, 1 hour recovery) or pretreated with the p38 inhibitor and irradiated

with UV light. Chromatin-associated proteins were extracted from cells, digested in-gel into

peptides and peptide samples were analyzed by LC-MS/MS.

b. The Spearman’s rank correlation was calculated to determine the experimental reproducibility.

c. Total cell lysates of U2OS cells were resolved by SDS-PAGE and proteins were detected with

the indicated antibodies.

d. NELFE dissociates from chromatin in a p38-dependent manner after UV light in HaCaT cells.

Chromatin protein fractions from differentially treated cells were resolved by SDS-PAGE and

subjected to western blotting with the indicated antibodies.

e. U2OS cells were exposed to UV light and left to recover for different time points. Total cell

lysates were resolved by SDS-PAGE and proteins were detected with the indicated antibodies.

 

Supplementary Figure 7a b

c

e

1000 3000

RN

A p

ol II

occ

upan

cy

bp-300

TSS Downstream

Pol II release ratio (PRR) =Downstream

TSSPO

LR2A

POLR

2B

POLR

2C

POLR

2D

POLR

2E

POLR

2G

POLR

2H

POLR

2I

POLR

2J

0

1

2

8.1e

−02

1.6e

−01

2.6e

−01

9.9e

−01

1.6e

−02

1.7e

−01

4.3e

−01

3.7e

−01

1.4e

−01

d

Untreated UV Untreated UV

RNA pol IIChIP-seq

GRO-seq(Williamson et al.)

RN

A po

l II r

elea

se ra

tio (P

RR

)

-4

0

4

8

2626 150470.8%

61929.1%

RNA pol II/NELFE targets

RNA pol II targetswith PRR up

4037 4130 2768

RNA pol IItargets

(U2OS)

NELFEtargets (HeLa)(Stadelmayer et al.)

chromosome organizationDNA recombination

protein localization to cytoplasmic stress granulecellular macromolecule catabolic process

DNA replication−independent nucleosome organizationchromatin silencing at rDNA

CENP−A containing chromatin organizationprotein heterotetramerization

protein modification by small protein conjugation or removalcentromere complex assembly

histone exchangenon−recombinational repair

gene silencing by RNADNA methylation on cytosine

histone H4−K20 demethylationdouble−strand break repair via nonhomologous end joining

RNA splicingRNA processing

DNA replication−dependent nucleosome organizationtelomere organization

negative regulation of hematopoietic progenitor cell differentiationtelomere maintenance

mRNA metabolic process

Fold enrichment

0.0 1.0 2.0

9.2e−028.4e−02

8.3e−025.4e−02

5.1e−024.2e−02

2.2e−022.0e−02

1.7e−021.5e−021.5e−02

1.2e−021.1e−02

7.8e−035.8e−03

5.4e−031.4e−03

9.5e−046.7e−04

6.2e−045.8e−04

2.5e−046.5e−05

fPhosphoproteomics - UV light increases phosphorylation of 538 sites

- 138 sites are phosphorylated in a p38-dependent manner- MK2/3 act downstream of p38 in response to UV light- RBPs, including NELFE, are phosphorylated by p38-MK2

Interactome analysis - 14-3-3 dimers bind to proteins phosphorylated by MK2- NELFE binds to 14-3-3 after UV light

Biochemistry / X-ray crystallography

- NELFE and 14-3-3 interact directly in a UV light- and p38-dependent manner

RNA pol II ChIP-seq - UV light leads to RNA pol II elongation

Chromatin proteome - DDR proteins are recruited to and excluded from chromatin after UV light- RBPs dissociate from chromatin in a p38-dependent manner

UV/untreatedp38i + UV/untreated

log 2(m

ean

SILA

C ra

tio)

Supplementary Figure 7: UV light exposure leads to transcriptional elongation

a. The bar plot shows the levels of RNA pol II subunits on chromatin quantified in untreated

U2OS cells and after UV light by SILAC-based quantitative MS. The error bars show the mean

and standard deviation of SILAC ratios quantified from three replicate experiments. Two sided

Student’s t test was used to assess the significance.

b. Schematic representation of the approach for the RNA pol II release ratio (PRR) calculation.

c. Comparison of PRRs calculated in untreated and UV light treated U2OS cells from two RNA

pol II ChiP-seq replicate experiments with PRRs calculated from GRO-seq data from the study

by Williamson et al. The lower and upper hinges represent the first and third quartiles (25th

and 75th percentiles, respectively). The line in the center of the box corresponds to the median

of the data range.

d. GO terms significantly enriched among genes with UV light-upregulated PRR. The bar plot

shows significantly overrepresented GO terms associated with genes containing upregulated

PRR compared to all RNA pol II bound genes. The significance of the enrichment of a specific

term was determined using a hypergeometric test.  P values were corrected for multiple

hypotheses testing using the Benjamini and Hochberg FDR.

e. NELFE target genes were extracted from the study by Stadelmayer et al. and overlapped with

genes displaying significantly increased PRRs after UV light exposure determined by RNA

pol II ChiP-seq. 70.8% of genes with increased PRR were found to be targets of NELFE in

HeLa cells.

f. A schematic overview of the methods and findings reported in this study.

 

Figure 1b

pChk1 (S345)

Vinculin

70 -

53 -

41 -

235 -

130 -

93 -

170 -

pChk2 (T68)

70 -

53 -

pp38 (T180/Y182)

53 -

41 -

30 -

Ponceau

53 -

41 -

30 -

Figure 1a

pp38 (T180/Y182)

pJNK1 (T183/Y185)

pChk1 (S345)

Ponceau

70 -

53 -

41 -

41 -

41 -

53 -

53 -

30 -

30 -

Supplementary Figure 8

pMK2 (T334)

Figure 4b

53 -41 -

30 -

Ponceau

NELFE

NELFE

53 -41 -

30 -

53 -41 -

70 -

FLAG M2

53 -41 -

30 -

Figure 4c

p38NELFE pulldown

MK2

NELFE input

GST (14-3-3)

pChk1 (S345)

Ponceau

53-

41-

70-

53-

41-

70-

Figure 4d

pChk1 (S345)

14-3-3 motif

GFP (NELFE) input

GFP (NELFE) pulldown

Ponceau

53-

41-

70-

53-

41-

70-

53-

41-

70-

70-

53-

93-

Vinculin

pMK2 (T334)

Figure 4e

53 -

41 -

53 -

41 -

53 -

41 -

53 -

41 -

235 -170 -130 -

235 -170 -130 -

Figure 4f

NELFE pulldown

Ponceau pulldown

pp38 (T180/Y182)

pMK2 (T334)

NELFE input

Ponceau input

- 53

- 41

- 70

- 53

- 41

- 70

- 53

- 41

- 70

53-

41-

70-

Vinculin

- 235- 170- 130

- 93

Figure 4g

NELFE pulldown

Ponceau pulldown

NELFE input

XPC

CSB

pp38 (T180/Y182)

Vinculin

pChk1 (S345)

53 -

41 -

70 -

53 -

41 -

70 -

53 -

41 -

70 -

235 -170 -130 -

93 -

235 -170 -130 -

93 -

235 -170 -130 -

93 -

53 -

41 -

70 -

Ponceau

GFP (NELFE)

GFP (NELFE)

93 -

70 -

53 -

41 -

93 -

70 -

53 -

41 -

GST (14-3-3)

70 -

53 -

41 -

pChk1 (S345)

53 -

41 -

Figure 5c

Ponceau

14-3-3 (pan)

53 -

41 -

30 -22 -18 -

53 -

41 -

30 -22 -18 -

53 -

41 -

30 -22 -18 -

14-3-3 (pan)

Figure 5g

Figure 6d

53 -

41 -

41 -

30 -

93 -

70 -

Ponceau

PCNA

NELF-E

MCM7

Ponceau

MCM7

NELFE

53 -

41 -

93 -

70 -

Figure 6e

p38

pMK2 (T334)pp38(T180/Y182)

Vinculin235 -

130 -

93 -

170 -

53 -

41 -

53 -

41 -

30 -

53 -

41 -

pMK2 (T334)

Figure S1a

Vinculin

pMK2

pChk2 (T68)

pChk1 (S345)

Ponceau

53 -

41 -

70 -

53 -

41 -

70 -

53 -

41 -

70 -

93 -

170 -130 -

Figure S1b

Figure S4a

NELFE pulldown

GST (14-3-3)

pMK2 (T334)

pp38 (T180/Y182)

NELFE input

Vinculin

130 -

93 -

41 -

53 -

53 -

41 -

53 -

41 -

53 -

41 -

53 -

Ponceau

HaCaT

NELFE pulldown

Ponceau (14-3-3)

pMK2 (T334)

pp38 (T180/Y182)

NELFE input

Vinculin

Ponceau

RPE-1

130 -

93 -

41 -

53 -

53 -

41 -

53 -

41 -

53 -

41 -

NELFE pulldown

Ponceau (14-3-3)

pMK2 (T334)

pp38 (T180/Y182)

NELFE input

Vinculin

Ponceau

HEK293T

130 -

93 -

41 -

53 -

53 -

41 -

53 -

41 -

53 -

41 -

Figure S4b

NELFE pulldown

Ponceau (14-3-3)

NELFE input

41 -

53 -

53 -

41 -

53 -

pMK2 (T334)

41 -

53 -

Vinculin

130 -

93 -

Figure S6c and S6d

Ponceau

41 -

53 -

70 -93 -

MCM7

NELFE

Figure S6e

93 -

70 -

53 -

41 -

Ponceau

MCM7

NELFE

Supplementary Figure 8: Uncropped scans of all western blots

 

Supplementary Table 1

List of siRNAs used in this study

Gene name Sequence 5’-3’

p38 gaagcucuccagaccauuu

MK2 ccgaaaucaugaagagcau

MK3 ccaaagauguugugaggaa

MK5 ggagaaagacgcagugcuu

NELFE 1 cagccaagguggugucaaa

NELFE 3’UTR acacugagguggaagcuuac

XPC gcaaauggcuucuaucgaa

CSB ccactgattacgagataca

Supplementary Table 2

List of oligos used in this study

Construct Sequence 5’-3’

NELFE S49/51A gacaggctgctctgctagtgcgcgtttgacaccac

NELFE S115A gtcatcatcagcagctatgctcctctggaacgg

NELFE S251A ccgttcagggaatgcatccgacctgcgga

Supplementary Table 3

Crystallization data collection and refinement statistics. Values in parentheses are for the highest

resolution shell.

14-3-3/NELFE Data collection statistics Beamline SLS PX III Wavelength (Å) 1.000 Space Group C 2 2 21 Unit Cell (Å) a = 79.29, b = 81.19, c = 81.05

α =90.00, β = 90.00, γ = 90.00 Resolution (Å) 46.47 - 2.70

(2.85 - 2.70) Observed reflections 97555 (14510) Unique reflections 7463 (1059) Redundancy 13.1 (13.7) Completeness (%) 100.0 (100.0) Rmerge 0.126 (0.821) <I/I> 18.4 (3.5) Refinement statistics Reflections in test set 757 Rcryst 17.2 Rfree 24.1 Number of groups Protein residues 234 Ions and ligand atoms 0 Water 4 Wilson B-factor 50.55 RMSD from ideal geometry

Bond length (Å) 0.012 Bond angles (°) 1.493 Ramachandran Plot Statistics

In Favoured Regions (%) 224 (97.39) In Allowed Regions (%) 6 (2.61) Outliers (%) 0 (0.00)

Supplementary Table 4

List of antibodies used in this study

Protein name Product number Origin Dilution

GFP sc-9996 Santa Cruz 1:2000

FLAG F1804 Sigma 1:2000

pp38 (T180/Y182) 9216 CST 1:1000

p38 8690 CST 1:1000

pMK2 (T334) 3007 CST 1:1000

pJNK 9255 CST 1:1000

pCHEK2 (T68) 2661 CST 1:1000

pCHEK1 (S345) 2344 CST 1:1000

Vinculin V9264 Sigma 1:1000

NELFE ABE48 Millipore 1:1000

14-3-3 (pan) 8312 CST 1:1000

POLR2A sc-899 Santa Cruz ChIP

pCTD (S2) 13499 CST 1:1000

XPC 14768 CST 1:1000

CSB sc-398022 Santa Cruz 1:1000

GST sc-138 Santa Cruz 1:2000

PCNA sc-56 Santa Cruz 1:1000

MCM7 3735 CST 1:1000

MCM6 sc-9843 Santa Cruz 1:1000

p14-3-3 motif 9601 CST 1:1000