RNase protection with the 1S-containing probe. (A) Design of the experiment. The antisense probe contained the 1S exon (filled rectangle), part of the second exon common to all b5R transcripts (open rectangle), and portions of the vector sequence (single lines marked V). After hybridization to a b5R mRNA, which contains 1S, the entire probe (except for the vector sequence) is expected to be protected from RNase digestion. In contrast, transcripts with the 1M exon will only partially protect the probe, with generation of a smaller protected species. (B) Electrophoretic analysis of protected fragments. A total of 10 μg of total RNA from HeLa (lane H), TE672 (lane T), induced or not induced K562 cells (lanes Kc and Ki on right panel), reticulocytes (lane R) or torulla yeast (lane Y), 2 μg of polyA⁺ RNA from noninduced K562 cells (lane Kc on left panel), 26 μg of total RNA from primary erythroblast cultures (lane E) were hybridized overnight with 40,000 dpm of 1S antisense probe. One half (left panel) or 1 μg (right panel) of the same RNA samples was hybridized to GAPDH antisense probe (bottom row). Hybridization and digestion conditions were as in the legend to Fig 2. Numbers on the left indicate molecular weight markers as in Fig 2. The asterisk at the right of the panels indicates the position of the undigested probe. Some probe remained undigested in the samples of the right panel. The arrowhead indicates the position of the fully protected probe, due to the presence of S-transcript in reticulocyte and erythroblast RNA. The partially protected probe of 89 nt is visible in all samples.