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Fig. 3. GFP-actin expression and distribution in lacrimal acini. (A) Western blots of lysates (100 µg protein/lane) from non-transduced and transduced lacrimal acinar cells developed in parallel using a polyclonal anti-actin antibody (left) and a polyclonal anti-GFP antibody (right) and goat anti-rabbit IRDye^TM 800-conjugated secondary antibody. Lane 1, rabbit lacrimal acinar cells without transduction; lane 2, rabbit lacrimal acinar cells transduced with Ad-GFP, MOI of 6; lanes 3-5, rabbit lacrimal acinar cells co-transduced with Ad-Tc-GFP-Actin and Ad-tTA at MOIs of 1.5, 3 and 6, respectively. (B) Co-transduced lacrimal acini fixed and processed as in Materials and methods to label actin filaments with rhodamine-phalloidin (Rho-Phall, red). GFP-actin fluorescence is shown in green while blue indicates nuclei labeled with DAPI. Most soluble GFP-actin is quenched by fixation/permeabilization. (C) Confocal fluorescence and DIC images of rabbit lacrimal acini co-transduced to express GFP-actin were acquired at 10.5 seconds intervals over 16 minutes. The top row shows GFP-actin fluorescence, the DIC image, and an overlay. GFP-actin fluorescence at intervals throughout the time-lapse sequence is shown in the second row. In images: bars, 5 µm; *, lumena; arrow, region of actin invagination; arrowheads, SVs.

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