Figure 4. Reactivity of trypsin fragment I with MPEG in cerebellum, SI(+) and SI(−) type-I IP₃Rs.

(A) Cerebellum microsomes (Cb, lanes 1 and 2) or microsomes from COS cells transfected with the SI(+) (lanes 3 and 4) or SI(−) (lanes 5 and 6) splice variants of the type-I IP₃R were incubated with 0.5 mM MPEG-20 for 5 min. The reaction was terminated with DTT and the samples were then trypsin-digested as described in the Experimental section. The samples were then electrophoresed on SDS/10% PAGE gels and immunoblotted with NT-1 Ab. Shifted bands are labelled with open circles. (B) Samples were as in (A) but processed on the same gel at the same exposure to illustrate the relative position of the doublet of bands seen in the cerebellum microsomes. (C) The same experiment as in (A) but using a 5 kDa MPEG derivative. (D) The indicated membranes were incubated with MPEG-20 (0.5 mM for 5 min). The molecular-mass shift in the full-length receptor was monitored by immunoblotting with CT-1 Ab.