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Blood, Vol. 108, Issue 3, 965-973, August 1, 2006
The Wingless homolog WNT5A and its receptor Frizzled-5 regulate inflammatory responses of human mononuclear cells induced by microbial stimulation
Blood Blumenthal et al.
108: 965
Supplemental materials for: Blumenthal et al
Files in this Data Supplement:
- Figure S1. Wnt5a expression in TLR2-transfected HEK293 cells is not further enhanced by stimulation with TNF-α or TLR2-agonists (PDF, 23.2 KB) -
HEK 293-wildtype (HEK-wt) and TLR2-transfected HEK293 (HEK-TLR2) cells were stimulated with M. avium (MOI 10), synthetic lipopeptide (Pam3CSK4), LPS or recombinant human TNF- (each 10 ng/ml), and expression of Wnt5a mRNA was analyzed. IL-8 release of HEK293 cells (24 h) in duplicate wells (means ± SD) was measured as a functional control. HEK293 cells were stably transfected with an expression vector for human TLR2 (FLAG-tagged-huTLR2, a kind gift of Dr. P. Nelson, Seattle, USA, subcloned into pREP9 (Invitrogen, Karlsruhe, Germany). A batch of stable transfectants (HEK-TLR2) was obtained after cell culture in DMEM containing 10% FCS, 1% penicillin/streptomycin (complete medium) and 1mg/ml G418 (Invitrogen) for two weeks. Expression of huTLR2 was verified by FACS analysis with anti-TLR2 and anti-FLAG mAb. HEK-TLR2 cells were maintained in complete DMEM medium containing 400 µg/ml G418. One representative experiment out of two is depicted.
- Figure S2. Detection of Wnt5a protein in Wnt5a-overexpressing fibroblasts (PDF, 20.2 KB) -
1x106 cells of NIH-3T3 fibroblasts transfected with LacZ, Wnt5a and Wnt132 were lysed in Lämmli Buffer. Aliquots were separated by SDS-PAGE and the proteins transferred to nitrocellulose by Western Blot. After incubation with anti-Wnt5a antibody (R&D Systems) blots were incubated with peroxidase conjugated donkey-anti-goat secondary antibodies (Dianova) as decribed previously 63. Visualization was performed by enhanced chemiluminescence (ECL, Amersham). The specific signal only detected in Wn5a overexpressing cells in the range between 32 and 48 kDa resembles the molecular weight of Wnt5a (calculation based on amino acid sequence without considering posttranscriptional modifications; SwissProt database 68) with approximately 42 kDa.
- Figure S3. Characterization of the anti-Fzd5 antiserum (PDF, 28.1 KB) -
a. Solid phase ELISA: The Fzd5-derived peptide (8 nM) was bound to ELISA plates, plates were incubated with the anti-Fzd5 antiserum (dilution 1/1000) in the presence of free Fzd5 derived peptide (competing peptide) up to a 1000 fold molar excess compared to the bound Fzd5 peptide. Binding of the antiserum that remained in the presence of the competing peptide was calculated. b. 20 ng of recombinant human Fzd5/Fc (R&D Systems) were transferred to nitrocellulose membrane by Western Blot after SDS Page. After incubation with the pre-immune serum (PI) resp. the anti-Fzd5 serum both at a 1/5000 dilution, blots were incubated with a peroxidase conjugated goat anti-rabbit secondary antibody (Dianova) as decribed previously 63. Visualization was performed by enhanced chemiluminescence (ECL, Amersham).
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