Supporting Online Material

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A Conserved p38 MAP Kinase Pathway in Caenorhabditis elegans Innate Immunity
Dennis H. Kim, Rhonda Feinbaum, Geneviève Alloing, Fred E. Emerson, Danielle A. Garsin, Hideki Inoue, Miho Tanaka-Hino, Naoki Hisamoto, Kunihiro Matsumoto, Man-Wah Tan, and Frederick M. Ausubel

Supporting Online Material

Materials and Methods

Feeding RNAi was carried out as described in (S1, S2). Control wild-type worms at the L4 stage were fed HT115 E. coli carrying the L4440 expression vector only, and compared to wild-type worms that were fed HT115 carrying L4440-derived vectors with sequence specific to pmk-1 or pmk-2. Progeny of these worms were grown on the HT115 plates until the L4 generation and then transferred to PA14 slow killing plates. For pmk-1 RNAi, pDK177 was constructed by amplifying a 480-bp fragment spanning exons 1 and 2 of B0218.3 (pmk-1) from genomic N2 DNA and subcloning into L4440. A second RNAi vector, pDK179, which expressed RNAi corresponding to sequences specific to pmk-1 exon 3, gave equivalent results to pDK177. Two different vectors carrying sequence specific for pmk-2 did not give an Esp phenotype, and the reported larval lethal phenotype observed for the pmk-2 deletion mutant (S3) was not seen with pmk-2 RNAi, although negative RNAi results are not necessarily informative. Polyclonal rabbit antibody to PMK-1 was produced against peptides corresponding to amino acids 356-368 (NH2�ISDFQKNVAFADE�COOH) of PMK-1. The anti-ACTIVE p38 pAb (Promega), which specifically recognizes the doubly phosphorylated TGY motif of activated p38 was used as the anti-phospho-p38 antibody. The E7 Greek Letter Beta-tubulin antibody (used as a loading control) was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA 52242.


S1. A. Fire, S. Xu, M. K. Montgomery, S. A. Kostas, S. E. Driver, C. C. Mello, Nature391, 806-811 (1998).

S2. A. G. Fraser et al., Nature408, 325-330 (2000).

S3. K. Berman, J. McKay, L. Avery, M. Cobb, Mol. Cell. Biol. Res. Commun.4, 337-344 (2001).