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Blood, Vol. 111, Issue 4, 2310-2320, February 15, 2008
Oncogenic association of the Cbp/PAG adaptor protein with the Lyn tyrosine kinase in human B-NHL rafts
Blood Tauzin et al.
111: 2310
Supplemental materials for: Tauzin et al
Lipid analysis (Figure S1)
Ultracentrifuged rafts were extracted with chloroform/methanol for 48h at 40°C, the glycerophospholipids hydrolyzed in NaOH and the sphingolipids analyzed by high performance thin layer chromatography as previously described 1. Metacrylate treatment of thin layer chromatography plates and cholera toxin B overlay was carried out as described in 2. Alternatively, cells were labeled with 14C-serine 3, separated by high performance thin layer chromatography and revealed by autoradiography. In vitro kinase assay (Figure S2)
Raft membranes were pelleted by ultracentrifugation, resuspended in kinase buffer 20 mM MOPS (pH 7.5), 5 mM MgCl2, 5 mM MnCl2, and 0.1 mM Na3VO4. Following a 30 min preincubation with increasing concentrations of PP2 or CGP76030, phosphorylation was initiated by adding 10 ⌠Ci of 32P- -ATP (Amersham, 5000Ci/mmol) for 30 min, and stopped with addition of 6× sample buffer and boiling with DTT. Phosphorylated proteins were separated on 10% SDS-PAGE. The gel was thoroughly washed to remove free 32P-ATP, dried and autoradiographed. Quantitation was carried out with a Typhoon reader (Amersham), using PhosphorImager and ImageQuant. REFERENCES 1. Arni S, Ilangumaran, S, van Echten-Deckert, G, Sandhoff, K, Poincelet, M, Briol, A, Rungger-Brandle, E, Hoessli, D,C. Differential regulation of Src-family protein tyrosine kinases in GPI domains of T lymphocyte plasma membranes. Biochem. Biophys. Res. Commun. 1996;225:801-807.
2. Muthing J, Cacic M. Glycosphingolipid expression in human skeletal and heart muscle assessed by immunostaining thin-layer chromatography. Glycoconjugate J. 1997;14:19-28.
3. Van Echten G, Birk R, Brenner-Weiss G, Schmidt RR, Sandhoff K. Modulation of Sphingolipid Biosynthesis in Primary Cultured Neurons by Long Chain Bases. J. Biol. Chem. 1990;265:9333-9339.
Files in this Data Supplement:
- Table S1. Lyn siRNA and PAG siRNA (PDF, 12 KB)
- Figure S1. ALK+ and Raji cell lines exhibit quantitatively similar, but
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(A) Directly stained raft (pooled fractions 3-5) sphingolipids (alkaline-treated raft
lipid sample), following charring and staining of the TLC plate. c: authentic lipids, 1: Raji; 2: SR786; 3: SupM2. GlcSer, glucosylceramide; LacSer, lactosylceramide; SM, sphingomyelin; GSL, glycosphingolipids. (B) 14C-serine labeling of cells, followed by subcellular fractionation of detergentresistant raft material and autoradiography. c, 1, 2 and 3, as in Fig. S1 A. (C) Overlay of the metacrylate-treated TLC plate (Fig. S1 A) with alkalinephosphatase conjugated cholera toxin B subunit. The CTX blot of Fig. S1 C corresponds to the inset of Fig. S1 A including GM1. c, 1, 2 and 3, as in Fig. S1 A.
- Figure S2. Kinase inhibitors inhibit the Lyn kinase in isolated B-NHL rafts (JPG, 64.8 KB)
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In vitro kinase assays with raft aliquots (containing equal amounts of Lyn) from Raji, DoHH2, Jeko-1 and VAL cells, following pretreatment (30 min) with increasing amounts of CGP76030 and PP2. Autoradiogram (left) and quantitation of the 80 kDa component and Lyn (53 and 56 kDa) phosphorylations (right). The results presented are representative of two independent experiments.
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