In this study, commercially available dynein and kinesin antibodies (BabCo, Berkeley, CA) were used to screen for immunoreactive motor proteins in testis tissue sections and in epithelial fragments. The cytoplasmic dynein antibody used was raised against the intermediate chain (IC74). Ki-nesin antibodies tested included those raised against kinesin heavy chain (SUK-4), kinesin light chain, kinesin-II, and a kinesin-related protein (HIPYR). Since preliminary data indicated that only the IC74 dynein antibody, the kinesin-II antibody, and the antibody to the kinesin heavy chain (previously immunolocalized to the manchette and trans-Golgi network ) produced significant immunofluorescence, the work reported here involves only the former two antibodies, for which testis immunostaining has not previously been reported. buy flovent inhaler
To explore further the possibility that motor proteins could be associated with the spermatid/junctional complexes, gelsolin, an actin-severing enzyme that has been used previously to remove actin filaments from permeabilized cells, was used to disrupt the junction plaques, and low-speed centrifugation was used to remove spermatid heads from solution. We reasoned that if motor proteins were indeed associated with junction plaques, then the amount of these motors in supernatants collected from spermatid/junction complexes in which the junction plaques were artificially disassembled should be increased relative to controls. Supernatants from gelsolin-treated and control preparations were compared, on immunoblots, for their reactivity with antibodies to the motor proteins.