Anti-Microbial Activity and Cell Binding are Controled by Sequence Determinants in the Anti-Microbial Peptide PR-39 Yvonne R. Chan, Margherita Zanetti,

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Anti-Microbial Activity and Cell Binding are Controled by Sequence Determinants in the Anti-Microbial Peptide PR-39 Yvonne R. Chan, Margherita Zanetti, Renato Gennaro, Richard L. Gallo Journal of Investigative Dermatology Volume 116, Issue 2, Pages 230-235 (February 2001) DOI: 10.1046/j.1523-1747.2001.01231.x Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 PR peptide variants demonstrate altered ability to bind SH3-containing fusion protein.125I-PR peptides were incubated with GST-lck SH3 protein, cross-linked with BS3 and analyzed by SDS–polyacrylamide gel electrophoresis. Bound proteins shift the size of the signal above baseline of 125I-PR peptide alone to approximately 40 kDa. 125I-N3 (lane 1), 125I-K3 (lane 2), 125I-PPRR (lane 3), 125I-L9W (lane 4), 125I-RPRP (lane 5), 125I-NRPR (lane 6), 125I-PR-39(15) (lane 7). Data are representative of three experiments. Higher molecular weight bands (98 kDa) reflect multimers that cannot be used to assess 1:1 PR peptide-SH3 binding and their signal was corrected for in final analysis. Journal of Investigative Dermatology 2001 116, 230-235DOI: (10.1046/j.1523-1747.2001.01231.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 PR peptide variants bind NIH 3T3 lysate with different affinities. NIH 3T3 lysates were added to high-binding enzyme-linked immunosorbent assay plates and blocked with 1% bovine serum albumin. Binding of biotinylated PR peptides was detected with streptavidin– alkaline phosphatase and measured by colorimetric assay of alkaline phosphatase substrate. Data are representative of four experiments. Journal of Investigative Dermatology 2001 116, 230-235DOI: (10.1046/j.1523-1747.2001.01231.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Syndecan-4 mRNA induction by PR peptides. Peptides were added to NIH 3T3 grown to confluency in 96-well plate. RNA from cells was transferred to nylon membrane and detected with an [α32P]deoxycytidine triphosphate-labeled syndecan-4 polymerase chain reaction primer. Densitometric analysis was done on the autoradiograph of triplicate determinations from two experiments. Signal was detected using a PhosphorImager and quantified in arbitrary units as all samples were analyzed on the same membrane. This value was then normalized to the amount of RNA placed on the membrane for comparison with other peptides. Journal of Investigative Dermatology 2001 116, 230-235DOI: (10.1046/j.1523-1747.2001.01231.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions