Figure 6. Relative kinetics of the binding of MPEG to fragment I and fragment III bands.
(A) COS cell microsomes from cells transfected with SI(+) (lanes 1–3) or SI(−) (lanes 4–6) type-I IP3R were incubated with 0.5 mM MPEG-20 for 15 s (lanes 2 and 5) and 5 min (lanes 3 and 6) before being trypsin-digested for 5 min. The digested membranes were electrophoresed on SDS/10% PAGE gels and immunoblotted with NT-1 Ab for detection of fragment I and with Frag3 Ab for fragment III. Shifted bands are indicated by open circles. (B) SI(+) microsomes were allowed to react with MPEG-20 for 15 s or 5 min and then treated with trypsin for 5 min as described in the Experimental section. The shifts in fragment I were detected with NT-1 Ab. In lane 4 the membranes were treated with trypsin for 5 min and then treated with a 10-fold excess of soybean trypsin inhibitor before reaction with MPEG-20 for 5 min. (C) The experiment was performed as in (B) but using MPEG-5.