JCS01403 Supplementary Material

Materials and Methods

Extracts of worm proteins were biotinylated as described previously (Gettner et al, 1995), except that crude homogenates were prepared by sonication.  Cleared lysates were incubated with 0.5 ml MH25 antibody (Francis and Waterston, 1991; Williams and Waterston, 1994) or anti-GFP antisera (Clontech), and then precipitated with Protein G sepharose beads (Amersham).  The precipitates were subjected to 8% SDS-PAGE under reducing conditions, then transferred to an Immobilon PVDF membrane (Millipore).  The blots were incubated with 1:5000 diluted HRP conjugated streptavidin (Chemicon), and binding was detected by chemiluminescence using the ECL system (Amersham).

Francis, G. R. and Waterston, R. H. (1991). Muscle cell attachment in Caenorhabditis elegans. J. Cell Biol. 114, 465-479.

Gettner, S. N., Kenyon, C. and Reichardt, L. F. (1995). Characterization of bPat-3 heterodimers, a family of essential integrin receptors in C. elegans. J. Cell Biol. 129, 1127-1141.

Williams, B. D. and Watersoton, R. H. (1994). Genes critical for muscle development and function in Caenorhabditis elegans identified through lethal mutations. J. Cell Biol. 124, 475-490.

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. No physical interaction between TSP-15 and PAT-3. Western blot analysis of biotinylated 1% Ttinton-X 100 or 1% Brij97 lysates of mixed-stage N2 or Ex[tsp-15::gfp] worms immunoprecipitated with anti-GFP or anti-PAT-3 antibody (MH25). Under reducing conditions, PAT-3 migrated at ~120 kD (Gettner et al, 1995) and TSP-15::GFP at ~57 kD, respectively.  TSP-15::GFP protein was not detected in a control lysate of N2 worms. Anti-GFP antibody failed to coimmunoprecipite integrins in both Triton-X 100 and Brij 97 lysates. Also, co-immunoprecipitation by anti-PAT-3 antibody did not show an association between TSP-15 and integrins.