Western blots of different cell and tissue preparations. (A) Normal and OHSt red cell membranes. Blots 1A, IB, and II were probed with rabbit polyclonal serum, and blot III was probed with mouse monoclonal. Blots IA, IB, and III show comparison of patient A-II-1 with healthy control, whereas blot II shows patient B-II-1. At the shorter ECL exposure time (blot IB), no signal can be seen in the “patient” lane (P), but at the longer exposure time (IA, II), further bands at higher molecular weight are evident in the “healthy” lane (N), which most likely represent oligomers of stomatin itself, since they are not evident in the patient lane. In blot III, the monoclonal antibody identifies further bands at slower migration (*), which probably represent nonspecific binding since the bands are present in both samples. (B) Probing of dilution series of normal red cell membranes with polyclonal antistomatin antibody. Serial dilutions of normal red cell membranes were run on a 12% polyacrylamide gel, blotted to nitrocellulose, and probed with the rabbit polyclonal antibody using a long ECL exposure time. At low dilutions, bands at 55 to 65 kDa are evident, as in blots IA and II seen in panel C. (C) Test of binding of polyclonal antibody to SLP1 and SLP2, in the form of glutathione-s-transferase (GST) fusion proteins expressed in Escherichia coli. Upper panel, Coomassie stain; lower panel, antistomatin antibody Western blot of an identical gel. Lanes 1 to 4 in both panels: E coli lysates containing isopropyl β-D-thiogalactopyranoside (IPTG)–induced fusion proteins (lane 1, GST-SLP1; lane 2, GST-SLP2; lane 3, GST alone; lane 4, GST-stomatin), (aa 144-288; lane 5, molecular weight markers; lane 6, normal red cell membranes; lanes 8-9, 1:50 and 1:100 dilutions of GST-stomatin [aa 144-288] sample used in lane 4). Letters indicate positions of protein bands of interest as follows: a, GST-SLP1; b, GST-SLP2; c, GST alone; d, GST-stomatin (aa 140-288); e, stomatin in normal red cell membranes; f, 1 in 50 dilution of stomatin GST (aa 140-288); and g, 1 in 100 dilution of same. The antibody binds only to GST-stomatin (aa 140-288) (bands d, f, g) and native stomatin in red cells (band e), and not to SLP1 or SLP2. (D) Subcellular fractionation of nonerythrocyte circulating cells from normal blood, probed with polyclonal antibody. Platelets, neutrophils, lymphocytes, and monocytes were isolated as described in “Patients, materials, and methods.” Purities of more than 90% were achieved for each cell type, with less than 0.1% contaminating erythrocytes. Subcellular fractions were isolated by centrifugation on a sucrose step gradient, as described in “Patients, materials, and methods.” In each cell type, all fractions except cytosol show a positive band at 32 kDa. (E) Shown are 4 samples of normal and 1 sample of OHSt liver (patient A-I-2) probed with polyclonal antibody. A predominant single band at 32 kDa is evident. (F) Association of stomatin protein with buoyant, cholesterol + sphingomyelin–rich, Triton-insoluble material purified from normal and OHSt red cell membranes. Red cell membranes were treated with cold Triton X-100 and subjected to sucrose gradient ultracentrifugation.³⁶  Fractions were aspirated and run on 12% SDS polyacrylamide gels and blotted to nitrocellulose. Left-most lanes reflect the lower part of the gradient (45% sucrose wt/vol), whereas the right-most reflect the upper part of the gradient (0% sucrose). The blots were probed with polyclonal antistomatin antibody (upper panels) and antiflotillin antibody (lower panels). The upper panels show that about half of the stomatin was associated with this buoyant raft material, while the remainder was not. The lower panels confirm, first, that flotillin is present in both normal and abnormal red cells, second, that flotillin is exclusively associated with rafts in both cell types, and third, that even in the presence of a deficiency of stomatin, rafts still exist in these abnormal cells.