Monitoring Protein Phosphorylationfor the Ligand Screens

Goal: sample diversity of cellular response to inputs Current Approach:

Multiplex Western blotting with mixtures of phosphospecific antibodies

Quantify ligand-induced changes in site-specific protein phosphorylation

Ligand Screens: B Cells

Single ligand screen completed Two phosphospecific antibody mixes for

quantification of 11 phosphoproteins Timecourse data on-line Poster: “Analysis of B Cell Single Ligand Data”

Robert Sinkovits and Dennis Mock Dual ligand screen experiments and analysis

in progress. See posters: “Dual Ligand Screen in Splenic B Cells”

Keng-Mean Lin, Madhusudan Natarajan, Robert Hsueh, and Heping Han “Dual Ligand Screen in Splenic B Cells (continued)”

Keng-Mean Lin, Madhusudan Natarajan, Robert Hsueh, and Heping Han

Ligand Screens: WEHI-231

Poised to initiate screen: characterization of cells, derivation of protocols, validation of phosphospecific antibodies completed

See poster:“Preparation and Characterization of WEHI-231 for the Ligand Screens”

Robert Hsueh, Keng-Mean Lin, and Heping Han

Ligand Screens: Cardiac Myocytes

Two new antibody mixes developed Some antibodies that worked well for B cells did not for

myocytes Added antibodies for myocyte specific phosphoproteins

Ligand dose determinations completed Dual ligand screen initiated. See posters:

“Isolation and Culture of Adult Mouse Cardiac Myocytes for Signaling Studies”

Timothy D. O'Connell, Yan G. Ni, Keng-Mean Lin, Heping Han, and Zhen Yan

“Ligand Screen in Mouse Adult Cardiac Myocytes”Yan G. Ni, Keng-Mean Lin, Heping Han, and Timothy D. O'Connell

Current Phosphospecific Antibody

Mixtures

B cells Myocytes

Mix 1 Mix 2 Mix 3 Mix 4STAT6 STAT3 STAT3 STAT5

p90RSK PKC μ Akt p70S6 Kinase

Akt NFkB p65 GSK 3α/β ERK

ERK JNK p38 MAPK Ribosomal S6

p38 MAPK Troponin I Myosin L Chain

IkBα Phospholamban

Each antibody is directed to a specific site of phosphorylation on proteinColor-coding highlights 8 different pathways currently monitored in each cell typeProspective: Can we assay more phosphoproteins?Add ribosomal S6 and myosin light chain to Mix 1Another mix for leukocytes: Could start with p70 S6 kinase and GSK 3 from Mix 3 & 4

Insulin ReceptorSignaling

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*

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*Antibody leukocyte mix

*Antibody in myocyte mix

*Good antibody not in mix yet

Antibody Database

– Tabulation of results from antibody testing by Western immunoblotting

– Now available on-line (go to Data Center, Resources)

– Easy to spot antibodies that yielded promising results (highlighted with red check marks)

– See poster:“AfCS Antibody Laboratory’s Database:Tabulation of Our Experience with Commercially Available Antibodies”Heping Han, Becky Fulin, Lonnie Sorrells, Ruth Levitz, and Susanne Mumby

Monitoring Protein Phosphorylation:Increasing Our Repertoire

1 of 7 phosphospecific antibodies we test is suitable for multiplex Western blotting of whole cell lysates

Additional promising antibodies to proteins in pathways not represented by antibody Mixes 1 & 2 Smad 2 (S465/467) STAT1 (Y701) PKC delta (T505) Total current possibilities for multiplex = 10

Capacity: 80 samples/week with two mixes Best case scenario

2 more antibody mixtures for multiplex Western blotting Doubles the gels+blots, decreases number of samples we can assay to 40/week

Additional antibodies not good enough to multiplex More cells required More gels/blots to process means less than 40 samples/week

An alternative, less labor intensive assay could be our salvation

Monitoring More Phosphoproteins:Alternative Assay

Multiplex fluorescent bead technology Potential for monitoring 100 different analytes per

well of 96-well plate Licensed by Luminex Bio-Rad’s system: Bio-Plex

Cytokines Phosphoproteins

AfCS and Bio-Rad collaborate on development and validation of phosphoprotein assays

Bio-Plex Phosphoprotein Assays

Three-way Collaboration

1. AfCS and Cell Signaling Technology AfCS suggests targets for novel antibodies CST produces peptide antigen and antibodies CST and AfCS test antibodies AfCS uses antibodies and CST sells them

2. CST and Bio-Rad Collaboration agreement pending Development of antibody pairs (capture and

phosphospecific) for Bio-Plex

3. Bio-Rad and AfCS Develop/validate antibody pairs for Bio-Plex Set up Bio-Plex in AfCS to monitor protein

phosphorylation

Benefits of 3-way Collaboration

AfCS: Increase number of phosphoproteins monitored =

more data for modeling effort No increase in number of cells required

CST and Bio-Rad: Company name and product exposure Increased sales

Signaling Community: More AfCS data available to draw from Increased number of validated antibody reagents

available on the market Demonstration of the utility of Luminex technology for

analysis of phosphoproteins