Aberrant signaling in the PI 3-Kinase/Akt and Raf/MEK/ERK pathways is associated with the formation of cancerous tumors, while interplay between these two pathways contributes to pharmacological resistance. The goal of this study was to measure the e� ects of MEK and PI 3-Kinase inhibitors on the phosphorylation of 43 di� erent kinases using antibody arrays as screening tools. In both A549 human lung adenocarcinoma and T47D human ductal breast cancer cells, EGF treatment resulted in increased Akt (S473) and ERK1/2 (T202/Y204,T185/Y187) phosphorylation. In A549 cells, the phosphorylation of Akt was inhibited by the PI 3-Kinase inhibitors AS 605240, LY 294002, and PI 103. ERK phosphorylation in T47D cells was completely inhibited by the MEK inhibitor PD 032590, but displayed resistance to PD 98059 and U0126. Instead, an increase in Akt phosphorylation was observed with U0126 treatment compared to the untreated and other inhibitor treated cells. These results demonstrate the utility of the Human Phospho-Kinase array for monitoring o� -target inhibitor responses on interdependent pathways. The dose response of inhibitors on ERK and CREB phosphorylation was measured using arrays and con� rmed using ELISA with excellent correlation. By employing a chemiluminescence detection method, no specialized equipment beyond what is typically used to collect Western blot data was required.

The Human Phospho-Kinase array is an economical alternative to traditional methods such as Western blot for screening changes in kinase phosphorylation. Both plate-based and membrane-based arrays required 3.5 hours of hands-on time, making this method far more time-e� ective than performing multiple Western blots. By employing a chemiluminescence detection method, no specialized equipment beyond what is typically used to collect Western blot data was required. Both arrays are su� ciently sensitive to measure changes in phosphorylation caused by both ligand and inhibitor treatment, and were shown to be comparable to ELISA. This method also allows for the facile evaluation of inhibitor selectivity to o� -target kinases.

Pro� ling Kinase Phosphorylation using Antibody ArraysGreta Wegner, Erin Eleria, Jackie Ernst, Amy James | R&D Systems, Inc., USA

ABSTRACT ASSAY PRINCIPLE

CONCLUSIONS

Figure 1. Induction and Inhibition of Kinase Phosphorylation in Lung Adenocarcinoma Cells. The A549 human lung adenocarcinoma cell line was untreated or treated with AS 605240, LY294002, or PI-103 at 5 µM for 3 hours, followed by treatment with 100 ng/mL EGF for 15 minutes. Images of the Proteome Pro� ler Human Phospho-Kinase membrane array and the corresponding histogram pro� les are shown.

Recombinant Human EGF was from R&D Systems (Catalog # 236-EG). Inhibitors were from Tocris, an R&D Systems Company: AS 605240 (Catalog # 3578), LY 294002 (Catalog # 1130), PI 103 (Catalog # 2930), PD 0325901 (Catalog # 4192), PD 98059 (Catalog # 1213), SL 327 (Catalog # 1969), U0126 (Catalog # 1144). Arrays were from R&D Systems: Membrane-based Proteome Pro� ler™ Phospho-Kinase Array (Catalog # ARY003B), Plate-based Proteome Pro� ler 96 Phospho-Kinase Array (Catalog # ARZ004). ELISA development kits were from R&D Systems: Phospho-CREB (S133) DuoSet® IC (Catalog # DYC2510), Phospho-ERK1(T202/Y204)/ERK2(T185/Y187) DuoSet IC (Catalog # DYC1018B). Antibodies were from R&D Systems: Anti-Human/Mouse/Rat Phospho-ERK1 (T202/Y204)/ERK2 (T185/Y187) A� nity Puri� ed Polyclonal Antibody (Catalog # AF1018), Anti-Human/Mouse/Rat Total ERK1/ERK2 Monoclonal Antibody (Catalog # MAB15761).

Figure 2. Induction and Inhibition of Kinase Phosphorylation in Breast Cancer Cells. The T47D human ductal breast epithelial cell line was untreated or treated with 10 µM PD 0325901, 20 µM PD 98059, 10 µM SL 327, or 10 µM U0126 for 2 hours, followed by treatment with 100 ng/mL EGF for 15 minutes. Images of Proteome Pro� ler Human Phospho-Kinase membrane arrays and the corresponding histogram pro� les are shown.

Figure 3. Comparing Array Data to Single Analyte ELISA and Western Blot in a MEK Inhibitor Screen. The T47D human ductal breast epithelial cell line was untreated or treated with 10 μM PD 0325901, 20 μM PD 98059, 10 μM SL 327, or 10 μM U0126 for 2 hours, followed by treatment with 100 ng/mL EGF for 15 minutes. A. Phosphorylation was measured using Western blot. Histogram pro� les obtained from pixel densities acquired from the Proteome Pro� ler Array were compared to optical densities obtained from the DuoSet IC ELISA for Phospho-ERK1/2 (T202/Y204,T185/Y187) (B) and Phospho-CREB (S133) (C).

Figure 4. MEK Inhibitor Dose Response Measurements. The T47D human ductal breast epithelial cell line was treated with di� erent concentrations of PD 0325901 for 2 hours, followed by treatment with 100 ng/mL EGF for 5 minutes. A. ERK1/2 phosphorylation measured using Western blot. B. Images of Proteome Pro� ler 96 wells. Histogram pro� les obtained from pixel densities acquired from the Proteome Pro� ler 96 Array or the Proteome Pro� ler Array were compared to optical densities obtained from the DuoSet IC ELISA for Phospho-ERK1/2 (T202/Y204,T185/Y187) (C) and Phospho-CREB (S133) (D), respectively.

For research use only. Not for use in diagnostic procedures.www.RnDSystems.com

MATERIALS & METHODS

RESULTS

Membrane ContentMembrane-based Array

Akt (S473) HSP27 (S78/S82) PRAS40 (T246)Akt (T308) HSP60 Pyk2 (Y402)

AMPK α1 (T174) JNK pan (T183/Y185, T221/Y223) RSK1/2/3 (S380)

β-catenin Lck (Y394) Src (Y419)Chk-2 (T68) Lyn (Y397) STAT2 (Y689)c-Jun (S63) MSK1/2 (S376/S360) STAT3 (S727)CREB (S133) p27 (T198) STAT3 (Y705)EGF R (Y1068) p38α (T180/Y182) STAT5a (Y694)eNOS (S1177) p53 (S15) STAT5a/b (Y694/Y699)ERK1/2 (T202/Y204, T185/Y187) p53 (S46) STAT5b (Y699)

FAK (Y397) p53 (S392) STAT6 (Y651)Fgr (Y412) p70 S6 Kinase (T421/S424) TOR (S2448)Fyn (Y420) PDGF Rβ (Y751) WNK-1 (T60)GSK-3 α/β (S21/S9) PLC γ1 (Y783) Yes (Y426)Hck (Y411)

Target Phosphorylation Site

Akt1 S473ERK1/ERK2 (T202/Y204, T185/Y187)GSK-3β S9JNK Pan Speci� c T183/Y185p38α T180/Y182p70S6K T421/S424Src Y416HSP60 ----

Well MapPlate-based Array

PI 3-Kinase Inhibitor Screen

MEK Inhibitor Screen

Array vs Western Blot & ELISA

Akt

p38α

Reference Spot

Src

ERK1/ERK2 GSK-3β

p70S6KJNK PAN

HSP60

Membrane ArrayMany Analytes, Select Samples

Array Membrane

Streptavidin-HRP,Chemiluminescence

Substrate

Kinase Antibody

Target Kinase

Detection AntibodyLight

Plate-Based ArraySelect Analytes, Many Samples

Phos

pho-

ERK1

/2 (

Pixe

l Den

sity x

103 )

Phospho-ERK1/2 (O.D.)

15

10

5

20

25

35

30

0 0

Untreated EGF

PD 0325901

PD 98059SL 327

U0126

Untreated

EGFPD 0325901

PD 98059

SL 327U0126

Untreated EGF

PD 0325901

PD 98059SL 327

U0126

0.2

0.4

0.6

0.8

1.0

Phospho-ERK1 (T202/Y204)ERK2 (T185/Y187)

Total ERK1/2

ArrayELISA

ArrayELISA

Phos

pho-

CREB

(Pixe

l Den

sity x

103 )

Phospho-CREB (O.D.)

4

8

12

20

16

0 0

0.6

0.3

0.9

1.2

1.5

1.8

Untreated EGF

Membrane-based ArrayELISA

Phos

pho-

CREB

(Pixe

l Den

sity x

103 )

Phospho-CREB (O.D.)

5

10

15

25

20

0 0

0.75

0.5

0.25

1.0

1.25

1.5

1.75

Phos

pho-

ERK1

/2 (P

ixel D

ensit

y x10

3 )

Phospho-ERK1/2 (O.D.)

30

20

10

40

50

60

0 0

Untreated EGF

50 nM5 nM

1 nM250 pM

Untreated

Akt (S473)

EGF 50 nM 5 nM 1 nM 250 pMERK1/2 (T202/Y204, T185/Y187)

0.5

PD 0325901

1.0

1.5

2.0Plate-based ArrayELISA

EGF + PD 0325901EGF + PD 0325901

50 nM5 nM

1 nM250 pM

PD 0325901

Phospho-ERK1 (T202/Y204)ERK2 (T185/Y187)

Total ERK1/2

Untreated EGF 50 nM 5 nM 1 nM 250 pM

Untreated

EGF

AS 605240,EGF

LY 294002, EGF

PI 103,EGF

Akt (S473)

EGF R (Y1086)

CREB (S133) RSK (S380)

ERK1/2 (T202/Y204, T185/Y187)

EGF R (Y1086) Akt (S473) CREB (S133)

Phos

phor

ylat

ion

(Pixe

l Den

sity x

103 )

5

10

15

20

0

UntreatedEGFAS 60524LY 294002PI 103

Untreated

EGF

PD 0325901,EGF

PD 98059,EGF

SL 327,EGF

U0126, EGF

ERK1/2 (T202/Y204, T185/Y187)

Akt (S473) STAT3 (S727) c-Jun (S63)

Phos

phor

ylat

ion

(Pixe

l Den

sity x

103 )

20

25

30

5

10

15

35

0

UntreatedEGF

PD 0325901PD 98059

U0126SL 327

Akt (S473)

EGF R (Y1086)

CREB (S133)STAT3 (S727)

c-Jun (S63)

ERK1/2 (T202/Y204, T185/Y187)

Dose Response

A.

A.

B.

C.

B.

C.

D.