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Blood, Vol. 106, Issue 2, 626-634, July 15, 2005
Defective Vav expression and impaired F-actin reorganization in a subset of patients with common variable immunodeficiency characterized by T-cell defects
Blood Paccani et al.
106: 626
Supplemental materials for: Paccani et al, Vol 106, Issue 2, 626-634
Files in this Data Supplement:
- Figure S1. Defective CD3ζ phosphorylation and ZAP-70 activation in T-CVID T-cells (JPG, 92 KB)
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(A) Immunoblot analysis of postnuclear supernatants of PBL from T-CVID patients (T-CVID3 and T-CVID4) and a representative healthy control. The filter was sequentially probed with antibodies specific for phospho-CD3 or activated ZAP-70 (phospho-Y493) and reprobed after stripping with anti-ZAP-70 antibodies as loading control. (B) Immunoblot analysis with antiphosphotyrosine antibodies of ZAP-70 specific immmunoprecipitates from postnuclear supernatants of PBL from T-CVID patients (T-CVID3 and T-CVID4) and two healthy controls. After stripping, the filter was reprobed with control anti–ZAP-70 antibodies. The migration of molecular mass markers is indicated.
- Figure 2S. Expression of signaling proteins in T-CVID T-cells (JPG, 41 KB)
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Immunoblot analysis of postnuclear supernatants of PBL from T-CVID patients (T-CVID1-4), one or two disease controls (dis.ctr1 and 2), and a representative healthy control (ctr; n = 3). The filters were probed with the indicated antibodies. Antiactin mAb was used as loading control. The migration of molecular mass markers is indicated.
- Figure S3. Lck activity is not impaired in T-CVID T-cells (JPG, 11 KB)
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Lck autophosphorylation in in vitro kinase assays of Lck-specific immunoprecipitates from peripheral blood leukocytes from patients T-CVID3 and T-CVID4 and from a healthy control. After the kinase reaction, samples were subjected to SDS-PAGE, transferred to nitrocellulose filters, and analyzed using a PhosphorImager. The filter was then probed with anti-Lck mAb as immunoprecipitation control (top). The data are presented as relative autophosphorylation levels (bottom).
- Figure S4. Preliminary characterization of the Vav1 deletion in the T-CVID patient harbouring two Vav immunoreactive bands (T-CVID1) (JPG, 61 KB)
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(A) Schematic representation of the Vav1 gene locus. Exons (E1-27) are indicated as boxes. The regions amplified by PCR are indicated by red bars. The extent of the putative deletion is indicated by the red dotted line. (B) PCR analysis of the region spanning Vav1 exons 1, 3-4, 5-7, and 27 in the genomic DNA from T-CVID1 and a representative healthy donor. A portion of the gene encoding RNA polymerase II was used as an independent control. The PCR products were resolved and quantitated by laser densitometry. The graph shows the levels of PCR products obtained using the patient’s DNA as template, expressed as percent of the PCR products obtained using DNA from the healthy control (taken as 100%; n = 3-7). For exons 1 and 27, the analysis was also carried out on the genomic DNA from patient T-CVID3 (one Vav immunoreactive band) as an additional control (n = 2).
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