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BioMed Research International
Volume 2014, Article ID 349783, 10 pages
http://dx.doi.org/10.1155/2014/349783
Research Article

A Purified Recombinant Lipopeptide as Adjuvant for Cancer Immunotherapy

1National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Zhunan, Miaoli 35053, Taiwan
2Graduate Institute of Immunology, China Medical University, Taichung 40447, Taiwan

Received 26 November 2013; Revised 15 January 2014; Accepted 31 January 2014; Published 11 March 2014

Academic Editor: Alberto Reis

Copyright © 2014 Ying-Chyi Song et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Supplementary Material

Supplementary Figure 1. One microliter of the polished tryptic fragments was mixed with 1μl of a saturated solution of α-ciano-4-hydroxycinnamic acid in acetonitrile/0.1% trifluoroacetic acid (1:3, vol/vol). One microliter of the mixture was placed on the target plate of a MALDI micro MX mass spectrometer (Waters, Manchester, UK) for analysis. 3 μg of purified lipopeptides were loaded onto the Agilent 1100 series HPLC equipped with a 2.1x 100 mm POROS R1/10 column (Applied Biosystems®). The mobile phase A consisted of 0.1% formic acid in Milli-Q water and mobile phase B consisted of 0.1% formic acid in 100% CAN. a linear gradient was started from 5% to 100% B over a 20-min period and continued for 5 min at 100% B. After additional washing with 100% B for 5 min. Then, a steady gradient was washed with 20% B and 80% Isopropanol for 20 min. Later, we used 100% Isopropanol for additional washing, and change the buffer to 100% B. Finally, the buffer system was in 5% A and 95% B in 10 min.

Supplementary Figure 2. These lipopeptides were purchased from GeneDireX (Nevada, USA). These compounds are mixture of R and S stereoisomer. The stereochemistry of the compounds are: N-Palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-glutaminyl-[S]-glutamyl-[S]-alanyl-[S]-lysine (Pam3CSQEAK) and S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-glutaminyl-[S]-glutamyl-[S]-alanyl-[S]-lysine (Pam2CSQEAK). BM-DCs were generated from wild-type (WT), TLR1-/- (TLR1 KO), TLR2-/- (TLR2 KO), or TLR6-/- (TLR6 KO) mice as described in the manuscript. The cultured BM-DCs (1 x 106/ml complete RPMI-10 medium) were stimulated with indicated lipopeptides (10 μg/ml) or LPS (0.1 μg/ml) for 24 h. The levels of TNF-α in the culture supernatant were measured by ELISA. LPS (TLR4 agonist), was used as positive control. LCM (complete RPMI-10 medium) was used as negative control. The results are expressed as the means + S.D. of the amount of cytokine.

  1. Supplementary Figures