Fig. 3. (A) Distribution of Bet3 in various subcellular fractions derived from rat liver. 25 µg of protein from the indicated fractions (PNS, postnuclear supernatant; TM, total membrane; M, microsome-enriched membrane; G, Golgi-enriched membranes; C, cytosol) was separated by SDS-PAGE and stained with Coomassie Blue R-250 (right panel) or transferred to filters and immunoblotted with antibodies against Bet3 (left panel) or GS28 (centre panel). (B) Bet3 is present predominantly in the cytosol. NRK cells were fractionated into total membrane (TM; odd lanes) and cytosol (C; even lanes) fractions. Equivalent fractions of TM and C were separated by SDS-PAGE, transferred to a filter and immunoblotted to detect Bet3 (lanes 1 and 2), GS28 (lanes 3 and 4), ß-actin (lanes 5 and 6) or ß-tubulin (lanes 7 and 8). Their distributions between TM and C fractions were quantitated as shown below the blot. (C) Bet3 is a peripheral membrane protein. Golgi-enriched membranes were extracted with PBS, 1 M KCl, 2.5 M urea, 150 mM sodium carbonate (pH 11.5), or 1% sodium deoxycholate (1% DOC), respectively. The resulting supernatants (S) and pellets (P) were separated by SDS-PAGE, transferred to a filter and immunoblotted to detect GS28 (upper panel) or Bet3 (lower panel).