Fig. 3. Subcellular fractionation of RBL and RBL-Syt IX⁺ cells. Cell homogenates derived from RBL (A,B,D,E) or RBL-Syt IX⁺ (C) cells were fractionated on continuous sucrose gradients as described under Materials and Methods. Fractions were collected from the top, subjected to SDS-PAGE and immunoblotted with anti-N-Syt IX (A,B), anti-C2A-Syt IX (C) or anti-G_i2 (D) antibodies as indicated. To monitor the distribution of internalized Tfn (E), RBL cells were serum starved for 1 hour followed by 1 hour of incubation with biotin-conjugated Tfn (20 µg/ml) at 37°C before fractionation. Fractions subjected to SDS-PAGE and immunoblotted with HRP-conjugated streptavidin, visualized by ECL and the intensities of the bands corresponding to biotin-Tfn were quantified by densitometry.