Generation of a myoJ-specific antibody and creation of myoJ-null cells. (A) Shown are Coomassie blue–stained gels of whole cell extracts prepared from WT (JH10) cells (lane 2) and one of the myoJ-null cell lines (lane 3), and Western blots performed using extracts from WT cells (lanes 4 and 7) and two independent myoJ-null cell lines (lanes 5, 6, 8, and 9) and probed with either the crude (lanes 4–6) or the purified (lanes 7–9) anti-myoJ antibody. Note that the disruption of the myoJ gene results in the loss of the ∼225-kD band present in WT cell extracts probed with the crude anti-serum (see arrow), which this is the only band present when WT extracts are probed with the purified antibody, and that this band is absent in myoJ-null cell extracts probed with the purified antibody. (B) The schematic at the bottom shows the design of the linear DNA fragment designed to disrupt the myoJ heavy chain gene. This fragment contains two segments of heavy chain coding sequence placed 5′ and 3′ of the THY selectable marker cassette, which confers on JH10 cells the ability to grow in media lacking thymidine. The schematic also shows the predicted change in the size of an internal EcoR1 fragment present within the myoJ chromosomal locus after a double crossover, homologous recombination event involving the linear disruption fragment, as well as the positions of probe A (which should detect the shift in the size of the internal EcoR1 from ∼2.7 kb in WT cells to ∼5.7 kb in knockout cells) and probe B (which, because it corresponds to the gap between the two fragments of myoJ coding sequence used in the disruption fragment, should not recognize the ∼5.7 kb EcoR1 fragment generated in knockout cells). The Southern blots at the top show that all of these predictions hold true when genomic DNAs from JH10 cells (lanes 1 and 4) and two independent myoJ-null cell lines (lanes 2, 3, 5, and 6) are hybridized with probe A (left) and probe B (right) (see arrows).