Fig. 2. Expression and localization of PKN-1 in muscle. (a) GFP expressed by the
pkn-1 promoter was observed in many types of muscle. We injected a mixture of pKK44 plasmid (5 kb of upstream putative promoter, the first and second exons, and the first intron of
pkn-1was cloned into pPD95.75, a gift from Dr. Fire) and a
lin-15 rescuing plasmid into
lin-15 (
n765) [temperature-sensitive multivulva (Muv)] worms. To obtain transgenic animals, we picked non-Muv worms at 25 C. In transgenic animals, GFP driven by the
pkn-1 promoter was observed in the pharynx including the pharyngeal muscle (upper panel), body wall muscle (lower left panel), and vulva muscle (lower right panel). The scale bar represents 10 um. (b) GFP-tagged PKN-1 is localized at dense bodies (arrowheads) and M lines (arrows). We prepared transgenic worms harboring an extrachromosomal array containing both a plasmid expressing GFP-tagged full-length
pkn-1 cDNA under the control of heat shock promoter (hsp) and a plasmid for the
rol-6 (roller) dominant marker. Worms were fixed by the Nonet method.11 Antibody staining with anti-GFP (Invitrogen A11122, 1:200 dilution) and MH35 (1:200 dilution; alpha-actinin) or MH42 (1:200 dilution; UNC-89) was performed as described previously.9 All images were captured at room temperature with a Zeiss confocal system (LSM510) equipped with an Axiovert 100M microscope using an Apochromat 63/1.4 oil objective in 2.5 zoom mode. The color balances of the images were adjusted with Adobe Photoshop. The scale bar represents 10 um.