1. FXM phospho-specific antibody mix 1

Introduction

Summary

Gene perturbation by RNAi: See posters from the Molecular Biology lab and the Macrophage Biology labs.

Ligand treatment:1x106 RAW 264.7 cells were plated in each well in a 6-well plate, cultured overnight, and serum starved for 1 hr before ligand treatment. Cells were treated with 100 nM C5a, 25 M UDP for C5a or UDP receptor

activation. For Fcreceptor cross-linking, unless indicated otherwise, cells were treated with 5 g/ml IgG2a for 30 min, then with 44 g/ml F(ab)’2. Cells were treated for

1, 3, 10 or 30 min at 37 degree before harvest.

Western Blot:Preparation of RAW 264.7 lysates for western blot analysis: PP00000168 Protein transfer: PP00000005 Western blot analysis for RAW 264.7: PP00000181

We completed an extensive screen for antibodies to use in our analyses of phosphorylatioin-state changes in response to stimulation by C5a, UDP and Fc ligation. Ligand-specific patterns of protein phosphorylation were observed in the RAW cell extracts. Nevertheless, the detected changes in phosphorylation-state for each ligand were limited. For example, of over forty phospho-proteins analyzed in our initial screen of antibodies, only changes in phospho-Akt (S473) immunoreactivity were reliably detected in C5a or Fcreceptor activated RAW cells. Based on these results and evaluations previously done in the antibody lab, we have selected about 10 phospho-specific antibodies to form three tentative FXM phospho-specific antibody mixes. Among which, the mix 1 is most promising. We have begun to examine FXM ligands-induced protein phosphorylation changes in cell lines where particular gene expression is knock-down via RNAi. Preliminary results from these studies are reported here.

Acknowledgements

Protein Phosphorylation Studies in RNAi-mediated Gene Knock-down Cells

Yan G. Ni, Lily Jiang, Tamara Roach, Robert Rebres, Iain Fraser and Joelle Zavzavadjian

Scientific Staff of the AfCS Cell Preparation and Analysis Laboratory

Methods

FXM Antibody Mix 1

Apparent MW Source

FAK (Y925) 125 CST3284

Pyk2 (Y402) 116 CST 3291

p-p90RSK (S380) 90 CST 9341

p-Akt (S473) 65 CST 9271

CamKII (T286) 52, 60Promega V1111

p-Erk (T202/Y204) 46, 44 CST 9101

p-p38MAPK (T180/Y182) 42 CST 9216

1. Phospho-protein responses in C5a, UDP or Fc receptor activated RAW 246.7 cells using FXM phospho-specific antibody mix 1. Un, untreated. Phosphorylation state changes are evident for Akt in C5a treated cells; and for p90RSK, Erk1/2 and p38MAPK in UDP treated cells. Anti-Pyk2 (Y402) antibody was not used in this experiment. For Fc receptor cross-linking, cells were treated with 20 g/ml IgG2a and 66 g/ml F(ab)’2.

Antibody target

MW ( kDa) Source Score

Antibody target

MW kDa Source Score

Akt (S473) 65 CST 9271 1p38 MAPK (T180/Y182) 42 CST 9216 1

Akt (T308) 60 CST 9275 4p40Phox (T154) 40 CST 4311 3

Btk (Y223) 77 CST 3531 3p70 S6 Kinase (T389) 68 CST 9205 3

CamKII (T286)

52, 60

Promega V1111 2/3

p70 S6 Kinase (T421/S424) 68 CST 9204 2

c-Cbl (Y731) 120 CST 3554 4

Pak1(S199/204)/Pak2(S192/197)

61-7 /68-74 CST 2605 3

cPLA2 (S505) 110 CST 2831 3/4

pan-Src (Y416) 60 CST 2101 4

creb (S133) 43 CST 9191 2/3 paxillin (Y118) 68BioSource 44-722 3

FAK (Y397) 125 BD 611806 3 PDK1 (S241)61, 69 CST 3061 2/3

FAK (Y576) 125 BS 44-652 3PDK1/2 (T373/376)

60, 68 CST 3065 3

FAK (Y925) 125 CST3284 2

PKCzeta/lambda (T410/403) 76 CST 9378 4

FKHR (T24) /FKHRL1 (T32)

68, 97 CST 9464 2

PLC3 (S1105) 150 CST 2484 4

Gab1 (Y307) 115 CST 3234 2 PLC3 (S537) 150 CST 2481 4

Gab1 (Y627) 110 CST 3231 4 PLC1 (Y783) 155 CST 2821 4

Gab2 (Y452) 98 CST 3881 4PLC2 (Y1217) 150 CST 3871 2/3

GSK3 / (S21/9)

46, 52 CST 9331 3 Pyk2 (Y402) 116 CST 3291 2

Lyn (Y507)53, 56 CST 2731 3 Pyk2 (Y881) 132

BioSource 44-620 3

MARCKS (S152/156)

39, 68-90 Chemicon 2 Shp-2 (Y542) 72 CST 3751 3

mTOR (S2448) 289 CST 2971 3 Shp-2 (Y580) 72 CST 3754 3

mTOR (s2481) 289 CST 2974 3 Ship-1 (Y1020) 145 CST 3941 3

Myosin light chain (S20) 19

Rockland 600-401-416 2/3 Syk (Y323) 70 CST 2715 4

VASP 52 CST 3111 3Syk (Y525/526) 70 CST 2711 4

Phospho-antibody surveyScores: 1=unambiguous phospho-response to FXM ligands, little background (in red)2=weak phospho-response to FXM ligands, extra bands (in green)3=no phospho-response to FXM ligands , prominent band at expected size4=no phospho-response to FXM ligands, no prominent band at expected size

No change in P-Akt response to UDP

0.0

2.0

4.0

6.0

8.0

0.00 10.00 20.00 30.00

Time (min)

Fold

cha

nge pL_UGIP

Gai2

Increase in P-Akt response to C5a

0.02.04.06.08.0

10.012.014.016.0

0.0 10.0 20.0 30.0

Time (min)

Fold

cha

nge

pL_UGIP

Gai2

Decease in peak p38 MAPK responses to UDP

0.0

1.0

2.0

3.0

4.0

5.0

0.0 10.0 20.0 30.0

Time (min)

Fold

cha

nge pL_UGIP

Gaq

Decrease in P-pyk2 responses to UDP

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

0.0 10.0 20.0 30.0

TIme (min)

Fold

cha

nge pL_UGIP

Gaq

Akt

RhoGDI

NS C5a

1’C5

a 3’

C5a

10’

UDP

3’NS NS C5

a 1’

C5a

3’C5

a 10

’UD

P 3’

NS NS C5a

1’C5

a 3’

C5a

10’

UDP

3’NS NS C5

a 1’

C5a

3’C5

a 10

’UD

P 3’

pL_UGIP+PTx

pL_UGIP-PTx

Gi2+PTx

Gi2-PTx

Akt

RhoGDI

NS C5a

1’C5

a 3’

C5a

10’

UDP

3’NS NS C5

a 1’

C5a

3’C5

a 10

’UD

P 3’

NS NS C5a

1’C5

a 3’

C5a

10’

UDP

3’NS NS C5

a 1’

C5a

3’C5

a 10

’UD

P 3’

pL_UGIP+PTx

pL_UGIP-PTx

Gi2+PTx

Gi2-PTx

2. Changes in protein phosphorylation states in the Gi2 (top and middle panels) or Gq (bottom panel) gene knock-down cells. Immunoreactivity of phospho-Akt, -p38MAPK or -pyk2 were detected with FXM phospho-specific antibody mix 1. The normalized fold changes of ligand-induced responses over responses in untreated samples were measured at each time point. Average responses from three western blots (using the same set of samples) were shown in the top and bottom panels. For pertussis toxin (PTx) treatment, cells were treated with 50 ng/ml PTx

at 37 degree for overnight before ligand treatment. These results were also replicated with samples prepared in the Dallas Cell lab.

2. Assessing changes of protein phosphorylations in gene knock-down cells

Enhanced p-Akt response to C5a in Gi2 knock-down cells

Reduced P-p38MAPK, P-pyk2 responses to UDP in Gq KD cells

The enhanced P-Akt response to C5a is PTx sensitive

We have evaluated over 40 phospho-specific antibodies for their responses to C5a, UDP and Fcg ligation. Of these, 13 phospho-specific antibodies were selected to form three tentative FXM phospho-specific antibody mixes. Only the antibodies for phospho-Akt and -p38 MAPK detected robust and consistent responses in cells treated with a single ligand.

We have examined changes of FXM-ligand induced protein phosphorylation subsequent to gene perturbation. In Gq gene knock-down cells, there were diminished phospho-pyk2 and –p38MAPK responses to UDP, supporting an essential role of Gq in mediating these responses. In contrast, in Gi2 gene knock-down cells, an unexpected increase in C5a induced Akt phosphorylation was observed. Similarly, the C5a –induced calcium responses were also increased in these cells. These data suggest that as yet undetermined compensatory changes are a main form of responses to Gi2 knockdown in these cells.

Audra Wendt, Jason Polasek, Katherine Hawes, Richard Davis, Melissa Kachura, David Quan and Carrie Wong for technical assistance; the antibody lab for excellent support and intellectual input; Dr. Paul Sternweis and the macrophage committee for guidance.