Fig. 3. RIN3 transfected into HeLa cells co-localizes with Rab5 but not EEA1. (A) HeLa cells were transiently transfected with RFP-RIN3 and cultured for 48 hours. The cells were fixed and labelled with antibodies to Rab5 (top) or EEA1 (bottom). The fluorescence of RFP-RIN3 (left) and Alexa-488 secondary antibody (middle) was visualized by confocal microscopy, and merged images of the two signals are displayed in yellow (right). Arrowheads indicate the co-localization of RIN3 and Rab5. (B) HeLa cells transiently expressing RFP-RIN3 and GFP-Rab5b were subjected to confocal microscopy, and the fluorescence of RFP-RIN3 (left) and GFP-Rab5b (middle) was visualized by confocal microscopy as described in (A). (C) HeLa cells transiently expressing the full-length (FL, left), N-terminal (N, middle) or C-terminal (C, right) form of RFP-RIN3 were subjected to confocal microscopy. (D) HeLa cells transiently expressing RFP-RIN1 (left) or RFP-RIN3 (right) were subjected to confocal microscopy. (E) Lysates from the transfected cells (RFP-RIN1, left; RFP-RIN3, right) were separated by SDS-PAGE and immunoblotted (IB) with an anti-RFP antibody. Scale bars, 10 µm.

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