RAW 264.7 two-ligand screen

1
Macrophage Ligand Screen Phosphoproteins Nichola
s Wong, Robert Hsueh, Robert Sinkovits, and
Heping Han The Alliance for Cellular Signaling
The Antibody Laboratory (University of Texas
Medical Center at Dallas), the Cell Preparation
and Analysis Laboratory (University of Texas
Medical Center at Dallas), and the
Bioinformatics and Data Coordination Laboratory
(University of California at San Diego)
RAW 264.7 two-ligand screen 

• Strategy to Monitor Protein Phosphorylation for
  the Macrophage Ligand Screen
• Cell Preparation and Analysis Lab Expose RAW
  264.7 cells to single and paired combinations of
  ligands
• Goal 23 ligands and their 253 paired
  combinations conduct each 3 times on different
  days
• Experimental design
• Expose cells to 4 single ligands and their 6
  possible combinations
• 4 time points 1, 3, 10, 30
• Extract cells with SDS-PAGE sample buffer
  (supplemented with inhibitors of proteases and
  phosphatases) including 3 cultures of untreated
  cells 43 samples per experiment
• Antibody Lab Assay 21 phosphoproteins by
  multiplex Western blotting
• Centrifuge cell lysates to remove viscous DNA
• Produce blots with quadruplicate pairs of Bio-Rad
  Criterion gel (26-well, 4-20 gradient)
• Process each pair of blots with one of 4 mixtures
  of site-specific, phosphosensitive antibodies
• Create images of blots with Storm 860 imager
• Quantify signal from 21 phosphoproteins with aid
  of ImageMaster 1D Elite software
• Transmit data to Bioinformatics Lab
• Bioinformatics Lab Process data
• Create time course graphs from phosphoprotein
  data
• Display graphs on web www.signaling-gateway.org

Summary of Phosphoprotein Responses to Single
Ligands
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Ligands italic cAMP underline calcium
response bold phosphoprotein
only Phosphorylation up reproducible
increase up? possible increase down
reproducible decrease