µ µ µ µ

µ

µ µ µ

0.0000001

0.000001

0.00001

0.0001

0.001

0.01

0.1

1

-5 -4 -3 -2 -1 0 1 2 3 4 5p-v

alu

e (

T-t

est

)

log2 fold-change to parental cell line

Lapatinib resistant lysates - DigiWest

Aurora A - phospho Thr288

Aurora A (AIK)

Aurora B (AIM1)

Caspase 7

Caspase 9

Caveolin-1

cdc2 (CDK1)

CDK6

Cyclin B1

Cytokeratin 8

DUSP6 (MKP3)

DUSP9 (MKP4)

EGFR

EGFR - phospho Tyr1068

EGFR - phospho Tyr845

EphA2

Erk1/2 (p44/42) - pT202/Y204

Her2

IRAK4 Phospho T345/S346

LATS2

MKK6

p21/Waf1/Cip1

p27(Kip1) (CDKN1B) - pS10

p27(Kip1) (CDKN1B)

p53

Pyk2 / FAK 2

RSK 1

RSK 1/2/3

Src - phospho Tyr416

STEP (PTPN5)

YAP - phospho Ser127

H29

2 P

are

nta

l Re

p 1

H29

2 P

are

nta

l Re

p 2

H29

2 P

are

nta

l Re

p 3

H29

2 R

esi

stan

t R

ep 1

H29

2 R

esi

stan

t R

ep 2

H29

2 R

esi

stan

t R

ep 3

0.000001

0.00001

0.0001

0.001

0.01

0.1

1

-10 -5 0 5

p v

alu

e (

T t

est

)

Log2 fold changes control vs. Lapatinib resistant

DigiWest KB Pulldown Analysis

EGFR

EphA2 pS897

Cyclin B1

EphA4

0.000001

0.00001

0.0001

0.001

0.01

0.1

1

-5 -4 -3 -2 -1 0 1 2 3

Log

(10

) p

-valu

e (

AN

OV

A)

Triplicate KB-PD (Contr and Lapatinib resistant) – Kinases

Lysate

Kinobeads 2. DigiWest

3. DigiWest1. MassSpec

H292 parental H292 resistant+ 1uM Lapa nib

-4

-3

-2

-1

0

1

2

3

4

EG

FR

PK

C

IKK

cdc2

(CD

K1

)

Au

rora

A (A

IK)

Ep

hA

2

LIM

K1

Au

rora

B (A

IM1

)

AS

K1

Ca

sein

kinase

1 e

psilo

n

Ep

hB

4

SIK

3

LR

RK

2

Ab

l2

IRA

K4

Pyk2

/ FA

K 2

LA

TS

2

RS

K 1

CD

K6

MK

K6

Ep

hB

3

Fyn

Ep

hB

6

Ep

hA

4L

og

2 f

old

ch

an

ge

s

KinoBead pulldown analysis: MS vs. DigiWest

DigiWest

Mass Spectrometry

0.0001

0.001

0.01

0.1

1

-5 -3 -1 1 3 5

p v

alu

e (

T t

est

)

Log2 fold changes control vs. Lapatinib resistant

DigiWest Lysate Analysis

DUSP6 (MKP3)

IRAK4 pT345/S346

Aurora A - pT288

DUSP9 (MKP4)

CDK6

Anderes Bild

P

P

PP

Phosphorylation

Predicted inhibition

Effect not predicted

Predicted activation

Downregulation

Upregulation

P

0.000001

0.00001

0.0001

0.001

0.01

0.1

1

-5 -4 -3 -2 -1 0 1 2 3Log

(10

) p

-va

lue

(A

NO

VA

)

Log2 fold change

Triplicate KB-PD (Contr and Lapatinib resistant) – Kinases

0.000001

0.00001

0.0001

0.001

0.01

0.1

1

-5 -4 -3 -2 -1 0 1 2 3Lo

g(1

0)

p-v

alu

e (

AN

OV

A)

Log2 fold change

Triplicate KB-PD (Contr and Lapatinib resistant) – Kinases

EGFR IKBKE

AURKA

EPHB3

EphA4Fyn

RPS6KA1

-4

-3

-2

-1

0

1

2

3

4

EG

FR

PK

C

IKK

cdc2

(CD

K1

)

Auro

ra A

(AIK

)

Eph

A2

LIM

K1

Auro

ra B

(AIM

1)

AS

K1

Ca

sein

kina

se 1

Eph

B4

SIK

3

LR

RK

2

Abl2

IRA

K4

Pyk2

/ FA

K 2

LA

TS

2

RS

K 1

CD

K6

MK

K6

Eph

B3

Fyn

Eph

B6

Eph

A4

Lo

g2 f

old

ch

an

ge

s

KinoBead pulldown analysis: MS vs. DigiWest

DigiWest

Mass Spectrometry

1E-08

0.0000001

0.000001

0.00001

0.0001

0.001

0.01

0.1

1

-10 -8 -6 -4 -2 0 2 4p-v

alu

e (

T-t

est

)

log2 fold-change to parental cell line

Lapatinib resistant KB pulldown - DigiWest

EGFR

EphA2 pS897

Cyclin B1Fyn

EphA2

IRAK4 pT345/S346

EGFR

DUSP6 (MKP3)

Aurora A pT288

DUSP9 (MKP4)

ERK1/2 pT202/Y204 p27(Kip1) pS10

CDK6

Aurora A - pT288

0.0000001

0.000001

0.00001

0.0001

0.001

0.01

0.1

1

-5 -4 -3 -2 -1 0 1 2 3p-v

alu

e (

T-t

est

)

log2 fold-change to parental cell line

Lapatinib resistant lysates - DigiWest

DUSP6 (MKP3)

DUSP9 (MKP4)

ERK1/2 pT202/Y204p27(Kip1) pS10

CDK6

p53

µ