Silver nitrate

Trifluoroacetic acid (TFA), α-cyano-4-hydroxycinnamic acid (CCA), acetonitrile (ACN) and dithiothreitol (DTT) were obtained from Sigma (St. Louis, MO, USA). Immobiline drystrips, ammonium persulfate, urea, agarose, glycerol, bromophenol blue, iodoacetamide (IAA), silver nitrate, 3-[(3-cholamidopropyl)- dimethylammonio]-1-propane (CHAPS), and N, N, N’, N’-tetramethylethylenediamine (TEMED) were from Amersham Pharmacia Biotech (Uppsala, Sweden). Acrylamide, Bis, Tris, glycine, SDS and SDS-PAGE protein standards were from Bio-Rad (Hercules, CA).

Assays with proteins extracted from cell lysates, immunoprecipitations and Western blot analyses were performed as described previously [43 (link)]. Otherwise, recombinant GST-Syk (BPS Bioscience, San Diego, CA) and GST-ezrin (previously described in [68 (link)]) were used. In vitro kinase assays were performed as described [43 (link)]. For the two-dimensional electrophoresis analysis, proteins were precipitated for 2 h in two volumes of acetone at −20°C, and resuspended in 8 M urea, CHAPS 4%, and thiourea 2 M. We used 18 cm IPG-strips (Amersham Biosciences) with linear pH range of 3–10 for the first dimension. Proteins were loaded on the IPG-strips and run in a Multiphor II apparatus (Amersham Biosciences). After focusing, a second migration was performed in 10% SDS-PAGE gel and proteins were stained with silver nitrate (Amersham Biosciences).