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. 2013 Jul;19(7):971–981. doi: 10.1261/rna.038638.113

FIGURE 2.

FIGURE 2.

Characterization of the debranching reaction. A splicing reaction was carried out in Prp16-depleted Cwc25-HA extracts (lane 1), and the spliceosome was precipitated with anti-HA antibody (lane 2). The spliceosome was incubated at 25°C for 1 h under various conditions: (A) 10 mM Tris-HCl, pH 8.8 and 4 mM MgCl2 alone (lane 3) or with 150 mM LiCl, NaCl, KCl, RbCl, CsCl, or NH4Cl (lanes 4–9); (B) 10 mM Tris-HCl, pH 8.8, alone (lanes 3,8,13) or with 4 mM MgCl2, CaCl2, MnCl2, and ZnCl2, in the absence (lanes 3–7) or presence of 150 mM KCl (lanes 8–12) or 150 mM NaCl (lanes 13–17); (C) 10 mM Tris-HCl, pH 8.8, 4 mM MgCl2, 150 mM KCl (lane 4), or 60 mM KPO4, pH 8.8, 4 mM MgCl2, and 30 mM KCl (lane 5), or 10 mM Tris-HCl, pH 8.8, 4 mM MgCl2, and 150 mM NaCl (lane 6), or 60 mM NaPO4, pH 8.8, 4 mM MgCl2, and 30 mM NaCl (lane 7). (R) 1/10 of reaction mixture, (SP) spliceosome, (KP) KPO4, (NaP) NaPO4.