Sci. STKE, 10 December 2002
The tables provide access to downloadable Microsoft Excel files representing the data discussed in Pelech and Zhang. In addition, the methods that were used to obtain these data are described below.
Table 1. Relative distribution of protein kinases in monkey organs. [Microsoft Excel Spreadsheet]
Kinetworks KPKS 1.0 analysis was performed on 600 μg of crude lysate protein from 14 different monkey organs. For each of 78 protein kinases, the relative amounts of the kinase in all 14 different monkey organs were calculated as a percentage of the ECL signal in counts per minute (CPM) from the organ that displayed the strongest immunoreactivity signal. For those organs where the target kinase was not detected (ND) on the immunoblots, the box is left intentionally blank. The actual CPM values that corresponded to 100% for each target kinase are provided in the fourth column. The mean of the "percent of maximal" values for each kinase across the 14 organs is provided along with the standard deviation (SD). The lower the mean percentage value and the higher the standard deviation, the more selectively a given protein kinase is expressed. PSTK, protein serine-threonine kinase; PYK, protein tyrosine kinase; PTYK, protein threonine-tyrosine kinase.
Table 2. Comparison of protein kinase expression in monkey and rat organs. [Microsoft Excel Spreadsheet]
Kinetworks KPKS 1.0 analysis was performed on 600 μg of crude lysate protein from the brain, heart, skeletal muscle, spleen, and testes from monkeys and from those tissues and the thymus from rats. For each of 78 protein kinases, the ECL signal in counts per minute (CPM) is shown; for those organs where the target kinase was not detected (ND) on the immunoblots, the box is left intentionally blank.
Materials and Methods
Cell and Tissue Preparation
Human Jurkat T cells were obtained from the American Type Culture Collection (Bethesda, MD), and the peripheral blood mononucleocytes and platelets were purified from the blood of human volunteers. Organs were dissected from 400 g male Wistar rats and a 20-year-old male Rhesus monkey immediately after death, frozen in liquid nitrogen, and stored at 80°C. Total cell- and tissue-lysates were prepared as described (1). Thawed organ samples were weighed and lysed in homogenization buffer at pH 7.2 [20 mM 3-(N-morpholino) propane sulfonic acid (MOPS), 0.5% Nonidet P40, 2 mM EGTA, 5 mM EDTA, 30 mM sodium fluoride, 40 mM β-glycerophosphate, 20 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonylfluoride, 3 mM benzamidine, 5 μM pepstatin A, 10 μM leupeptin]. Four ml of homogenization buffer was used per 1 g of tissue. Tissues were then sonicated for 15 s and ultracentrifuged in a Beckman TL100 ultracentrifuge (100,000 g, 30 min, 4°C). The protein concentration in the supernatant fraction was determined by the method of Bradford (2), and the extracts were stored at 80°C.
Kinetworks Western Blot Analysis
Proteins from thawed cell- or tissue-lysates (300 μg/gel) were resolved on two mini-sodium dodecyl sulfate polyacrylamide gel eletrophoresis (SDS-PAGE) gels, according to the method of Laemmli (3). The Kinetworks KPKS 1.0 immunoblotting analysis involved probing with mixes of 75 primary antibodies from commercial sources validated at Kinexus for reliable immunoblotting performance. Each mixture was applied into a separate lane of a 20-lane multiblotter (Immunetics, Cambridge, MA). Detailed protocols for the Kinetworks analyses can be found at the Kinexus Bioinformatics Web site (http://www.kinexus.ca). The immunoblots were developed with enhanced chemiluminescence (ECL) Plus reagent (Amersham Pharmacia, Piscataway, NJ), and signals were then captured by Fluor-S MultiImager and quantified using Quantity One software (Bio-Rad, Hercules, CA).
Citation: S. Pelech, H. Zhang, Plasticity of the kinomes in monkey and rat tissues. Science's STKE (2002), http://www.stke.org/cgi/content/full/sigtrans;2002/162/pe50.