Fig. 1. Kette associates with membranes. (A) Schematic view of the Kette protein; hydrophobic domains are indicated. Different antisera were generated. N, N-terminal domain, amino acids 1-374; the M-serum directed amino acids 375-907 is not indicated; C, C-terminal domain, amino acids 908-1126; P, peptide antibodies, amino acids 652-666. (B) Western blot analyses of protein extracts prepared from wild-type, rhoGAL4/UAS-Kette full-length or rhoGAL4/UAS-Kette^myc embryos were probed either with the anti-Kette P antiserum or anti-Myc antibodies (Mab 9E10) as indicated. The Kette protein is 120 kDa. Anti-Myc antibodies recognize a similar sized protein confirming the specificity of the antisera. (C) Differential centrifugation reveals that Kette is located primarily in the cytosol. Western blots were probed with anti-Kette antisera (top), anti-actin antibodies (middle) and anti Na⁺/K⁺ ATPase antibodies (bottom) to monitor a typical transmembrane protein. The different lanes show: total, total protein extracts of S2 cells; PNS, supernatant following 1,000 g centrifugation. The supernatant was subjected to 25,000 g centrifugation and subsequently to 200,000 g centrifugation. The pellet was resuspended in either PBS at high pH or 1% NP-40 and subjected to 200,000 g centrifugation (S, supernatant; P, pellet). For each lane, equal amounts of total proteins were loaded on SDS-PAGE. (D) Post-nuclear supernatant (PNS) of wild-type embryos was subjected to equilibrium sedimentation on a discontinuous sucrose density gradient. Only small amounts of the Kette protein were detectable in the membrane fraction, while most of the Kette protein remains in high dense sucrose containing cytosolic proteins. Western blot analysis was performed using anti-Kette antisera (top) and anti Fas3 antibodies (bottom) to monitor a typical transmembrane protein. Equal amounts of total proteins in the post nuclear supernatant (PNS), membrane and cytosolic fraction were loaded for SDS-PAGE.