Figure 2. Muscle mRNA-tagging. A. Anti-FLAG antibody staining of a worm expressing myo-3p::FLAG::PAB-1 showing specific expression in striated muscle. bwm; body wall muscle, anterior is to the left. B. Western blotting analysis of immunoprecipitation of FLAG::PAB-1 (black arrow) from cell-free extracts. Western blots were stained with anti-FLAG antibodies. Samples 1 and 2 are extracts prepared from wild-type and FLAG::PAB-1-expressing animals, respectively. Lysate refers to the cell free lysate, super refers to the supernatant after immunprecipitation of FLAG::PAB-1, and eluate refers to immunoprecipitated material. Red arrows denote the light and heavy chains of the anti-FLAG antibodies used in the immunoprecipitation. The eluate lanes contain 1.6x more sample than the lysate and supernatant lanes. Approximately 55% of the FLAG:: PAB-1 present in the lysate is immunoprecipitated in the eluate sample. C. Dot blot analysis of muscle- and germ line-specific genes. Lane 1 is a 250 ng poly(A) mRNA standard. Lanes 2 and 3 contain RNA that was co-immunoprecipitated from mixed-stage wild-type and FLAG::PAB-1-expressing animals, respectively. The left blot was hybridized with an unc-54 probe, which is specifically expressed in body wall muscles. The blot on the right was hybridized with a gld-1 probe, which is expressed specifically in the germ line. Comparison of the amount of unc-54 RNA in the immunoprecipitated sample with that from the cell lysate shows that about 2% of unc-54 was co-immunoprecipitated with FLAG::PAB-1.