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. 2013 Jun 15;126(12):2704–2717. doi: 10.1242/jcs.122242

Fig. 4.

Fig. 4.

Characterization of mCherry-Rab3D expressed in LGACs. (A) LGACs co-transduced with Ad mCherry-Rab3D and Adeno-X Tet-On® were cultured overnight with 0.1 µg/ml doxycycline (Dox +) or not (Dox −); LGACs transduced with Adeno-X Tet-On® alone were cultured overnight with 0.1 µg/ml doxycycline (Tet-On® only). All LGACs were lysed with non-denaturing lysis buffer. Lysate was pre-cleared as in the Materials and Methods, and analyzed by western blotting using primary anti-Rab3D rabbit antiserum and mouse anti-mCherry monoclonal antibody, as well as secondary IRDye®800-conjugated goat anti-rabbit (green in the overlay image) and IRDye®700-conjugated goat anti-mouse (red in the overlay image) antibodies. Blue arrowhead, mCherry-Rab3D; green arrowhead, endogenous Rab3D. The expression level of mCherry-Rab3D was 5-fold higher than endogenous Rab3D. (B) LGACs expressing mCherry-Rab3D and non-transduced LGACs were fixed, permeabilized and labeled with primary rabbit anti-Rab3D serum, secondary Alexa Fluor®-488-conjugated donkey anti-rabbit antibody and Alexa Fluor®-647-conjugated phalloidin. White arrow, colocalization between red and green; *, lumena. Scale bars: 5 µm. (C,D) Immunoprecipitation (IP) was performed on lysates of LGACs co-expressing mCherry-Rab3D and hpIgR-EGFP as described in Materials and Methods. Rabbit anti-Rab3D serum was used to immunoprecipitate mCherry-Rab3D, and pre-immune rabbit serum was used as a control (C). Goat anti-hSC antibody was used to immunoprecipitate pIgR-EGFP, and pre-immune goat serum was used as control (D). The immunoprecipitated mCherry-Rab3D and hpIgR-EGFP were detected by western blotting, using mouse anti-mCherry, mouse anti-GFP and goat anti-hSC primary antibodies, as well as IRDye®700-conjugated goat anti-mouse (red in overlay), IRDye®800-conjugated donkey anti-goat or goat anti-rabbit (green in overlay) secondary antibodies. This image is representative for western blotting results from three preparations. Green arrowhead, hpIgR-EGFP; blue arrowhead, mCherry-Rab3D; red arrowhead, rabbit immunoglobulin heavy chain.