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Blood, Vol. 109, Issue 5, 2032-2039, March 1, 2007
ZAP-70 enhances B-cell–receptor signaling despite absent or inefficient tyrosine kinase activation in chronic lymphocytic leukemia and lymphoma B cells
Blood Gobessi et al.
109: 2032
Supplemental materials for: Gobessi et al, Vol 109, Issue 5, 2032-2039
Files in this Data Supplement:
- Figure S1. Analysis of BCR-induced phosphorylation of Tyr319 in ZAP-70 and Tyr352 in Syk in purified CLL B cells (JPG, 54 KB)
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Immunoblotting analysis is shown for 10 ZAP-70+ (G141, G42, G143, G34, G43, G122, G133, G4, G88, and G155) and 4 ZAP-70– (G101, G138, G104, and G140) CLL B-cell samples. Jurkat T cells and BJAB B cells were used as controls for ZAP-70 and Syk phosphorylation, respectively. Cells were stimulated with anti-IgM or anti-CD3, as indicated. The expected positions of the ZAP-70, Syk, and SykB proteins are indicated by arrows.
- Figure S2. Expression of ZAP-70 and Syk in stable BJAB transfectant clones (JPG, 19 KB)
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(A) Relative amounts of ZAP-70 and Syk mRNA in 3 clones transfected with the pcDNA3–ZAP-70–Myc construct. Diagrams represent gene fragment analysis of ZAP-70 and Syk RT-PCR products obtained from total RNAs of the BJAB stable transfectant clones, as previously described.19 The ZAP-70/Syk mRNA ratios are indicated inside each diagram. (B) Immunoblotting analysis of ZAP-70 protein levels in parental BJAB B cells, ZAP-70+ BJAB clones (clones A, D, and G), and Jurkat T cells. Membranes were reprobed with anti– -actin to ensure equal loading (lower panel).
- Figure S3. Ectopic expression of Lck in BJAB B cells does not induce phosphorylation of Tyr319 in ZAP-70 following IgM ligation (JPG, 14.4 KB)
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Parental and ZAP-70+ BJAB B cells were transiently transfected by electroporation with the pCEP4-Lck construct (kindly provided by M. Sefton, Salk Institute, La Jolla, CA) or with the pCEP4 empty vector, as a negative control. Transfected cells were stimulated with anti-IgM for 3 minutes or were left unstimulated. Immunoblotting analysis was done with phospho–ZAP-70Tyr319/SykTyr352 antibody (upper panel) and with anti-Lck antibody (Cell Signaling Technology; lower panel). The last lane was charged with a mixture of total cell lysates from BJAB parental B cells and Jurkat T cells that had been stimulated with anti-IgM or anti-CD3, respectively, as markers for phosphorylated Syk and phosphorylated ZAP-70 (indicated by arrows).
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