|
|
Blood, Vol. 111, Issue 4, 2132-2141, February 15, 2008
Disease-associated CIAS1 mutations induce monocyte death, revealing low-level mosaicism in mutation-negative cryopyrin-associated periodic syndrome patients
Blood Saito et al.
111: 2132
Supplemental materials for: Saito et al.
Files in this Data Supplement:
- Table S1. Primers and annealing temperature for allele-specific PCR (PDF, 115 KB) -
Allele-specific PCR was performed using 44-88 ng of genomic DNA, 4.125 pmolof allele-specific PCR primer for mutations found in mosaic patients and the corresponding reverse primer, 2.2 nmolof each of the four types of dNTPs, and 0.5 U of AmpliTaq-Gold DNA polymerase (Applied Biosystems) in a total volume of 11 µl. The PCR program was set to initial denaturationat 95°C for 5 minutes, followed by 32 cycles of amplification with 25 seconds of denaturationat 95°C, 30 seconds of annealing at the indicated temperature, and 30 seconds of elongation at 72°C, followed by additional elongation for 7 minutes at 72°C.
- Figure S1. Inhibition of IL-1 âsecretion by a caspase-1 inhibitor YVAD-fmk (PDF, 34 KB) -
PBMCs from patient 4 or a healthy control were incubated with or without YVAD-fmk (100 µM) for 30 minutes, and then with cLPS (10 ng/ml). After the indicated times of incubation, supernatants were collected and IL-1  concentration was determined by ELISA (eBioscience). Data is representative of 2 independent experiments.
- Figure S2. Enrichment of CIAS1-mutated monocytes in (A) patient 8, and (B) patient 9 (PDF, 150 KB) -
Left panel: flow cytometry analysis of PBMCs from each patient stimulated with cLPS for 2 hours. Right panel: chromatograms of the CIAS1 gene at position 790 (patient 8) or 919 (patient 9) from the indicated cell populations.
|
|
