2009-science-AIM2-supp.pdfVIP

Corrected 20 February 2009 /cgi/content/full/1169841/DC1 Supporting Online Material for HIN-200 Proteins Regulate Caspase Activation in Response to Foreign Cytoplasmic DNA Tara L. Roberts, Adi Idris, Jasmyn A. Dunn, Greg M. Kelly, Carol M. Burnton, Samantha Hodgson, Lani L. Hardy, Valerie Garceau, Matthew J. Sweet, Ian L. Ross, David A. Hume, Katryn J. Stacey* *To whom correspondence should be addressed. E-mail: k.stacey@.au Published 8 January 2009 on Science Express DOI: 10.1126/science.1169841 This PDF file includes: Methods Figs. S1 to S11 References Corrected 20 February 2009: The corrected SOM file includes details of the anti- p202(S-19) antibody on pages 4 and 5. Supporting online material. METHODS Cells and Cell Culture. Cell lines used were RAW264.7, NIH3T3, and J774, purchased from American Type Culture Collection, and RAW264.7 stably expressing p202 with a C-terminal V5 tag. Plasmid for the latter cell line was prepared by PCR amplification of full length coding sequences of p202a (1) and insertion into pEF6-V5/HisTOPO (Invitrogen). RAW264.7 cells were stably transfected with this plasmid. p202 is antiproliferative, and consequently can only be expressed at modest levels. Bone marrow-derived macrophages (BMM) were prepared and cultured as described (2) from BALB/c, C57BL/6, NZB mice or TLR9-/- mice backcrossed 12 times onto C57BL/6 background. NZB mice were supplied by Kew animal house (Walter and Eliza Hall Institute, Australia). TLR9-/- mice were obtained from Shizuo Akira, Research Institute for Microbial Diseases, Osaka University. Animal work was done under approval and in accordance to guidelines from the University of Queensland Animal Ethics Committee. RAW264.7 and J774 cells were cultured in RPMI1640 medium (Invitrogen) containing 5% foetal calf serum (FCS), 2mM L-glutamine, 20U/ml penicillin, and 20μg/ml streptomycin. NIH3T3 cells were cultured in DMEM medium (Invitrogen)