The supernatant (S) and microtubule pellet (P) were immunoblotted for dynein intermediate chain (dyn), p150, or -tubulin (tub)

The supernatant (S) and microtubule pellet (P) were immunoblotted for dynein intermediate chain (dyn), p150, or -tubulin (tub). for the study of liver disease. As predicted, canalicular delivery of all proteins tested was impaired in ethanol-treated cells. Unlike in control cells, transcytosing proteins were observed in discrete sub-canalicular puncta en route to the canalicular surface that aligned along acetylated microtubules. We further decided that this stalled transcytosing proteins colocalized with dynein/dynactin in treated cells. No changes in vesicle association were observed for either dynein or dynactin in ethanol-treated cells, but significantly enhanced dynein binding to micro-tubules was observed. From these results, we propose that enhanced dynein binding to microtubules in ethanol-treated cells prospects to decreased motor processivity resulting in vesicle stalling and in impaired canalicular delivery. Our studies also importantly show that modulating cellular acetylation levels with clinically tolerated deacetylase agonists may be a novel therapeutic strategy for treating alcoholic liver disease. and mark selected bile canaliculi. 10 m. B WIF-B cells were treated with 50 mM ethanol (EtOH) for 72 h. APN was constantly basolaterally labeled for 45 min and cells MK-3697 processed for immunodetection of trafficked (tr) APN and constant state (ss) dynein (dyn) (and point to regions of colocalization. 10 m Antibody trafficking in live cells To monitor transcytosis, cells were basolaterally labeled with antibodies specific to APN (1:50), 5NT (1:100) or myc (1:250; to label pIgA-R) for 20 min at 4 C. Cells were washed 3 times for 2 min on ice and then reincubated with prewarmed total medium. Antibody-antigen complexes were chased for 0, 45, or 90 min at 37 C and processed for immunofluorescence labeling. For the colocalization experiments, APN at the basolateral surface was constantly antibody labeled (1:100) for 45 min at 37 C. Cells were washed 3 times for 2 min with prewarmed total medium and processed for epifluorescence imaging. To quantitate the relative distributions of the transcytosing proteins, random fields from each slide were visualized by epifluorescence and digitized MK-3697 as explained. From micro-graphs, the average pixel intensity of selected regions of interest placed at the bile canalicular or basolateral surface of the same WIF-B cell were MK-3697 measured using ImageJ Measure ROI tool (National Institutes of Health) [14, 15]. The averaged background pixel intensity was subtracted from each value, and the ratio of bile canalicular to basolateral fluorescence intensity was decided. To monitor canalicular protein trafficking in VL-17A cells, pIgA-R expressing cells were labeled at 4 C for 20 min with anti-myc antibodies and washed as explained above. Antibody-antigen complexes were chased for 0 or 15 min at 37 C. To quantitate the relative protein trafficking levels, random fields Rabbit Polyclonal to GPR152 from each slide were visualized by epifluorescence and digitized. From micrographs, MK-3697 the number of cells containing intracellular labeling vs. total number of pIgA-R-expressing cells was counted and a percent was calculated. Western blotting Laemmli samples were prepared and boiled for 3 min [16]. Proteins were electrophoretically separated using SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies specific to -tubulin (1:7,500), acetylated–tubulin (1:2,000), dynein (1:1,000), or p150 (1:10,000). Immunoreactivity was detected using enhanced chemiluminescence (PerkinElmer, Crofton, MD). Relative protein levels were determined by densitometric analysis of immunoreactive bands and normalized to total -tubulin levels. Cell fractionation WIF-B cells produced on 10-cm dishes were detached with trypsin for 2 min at 37 C and pelleted by centrifugation. Cells were resuspended in 5C8 ml ice-cold swelling buffer (1 mM MgCl2, 1 mM DTT, 1 mM EDTA) and incubated 5 min on ice. Cells were pelleted by centrifugation and resuspended in 500 l of 0.25 M sucrose, 3 mM imidazole, pH 7.4 with added protease inhibitors (2 g/ml each of leupeptin, antipain, PMSF and benzamidine) and Dounce-homogenized with a tight fitting pestle for 20 strokes. The homogenate was centrifuged at 1,000 g at 4 C for 5 min to prepare a post-nuclear supernatant (PNS). The PNS was centrifuged at 20,000for 20 min at 4 C to prepare a heavy membrane population. The resultant supernatant was then centrifuged at 80, 000for 60 min at 4 C to prepare cytosolic and light membrane fractions. The fractions were immunoblotted for dynein, p150, and -tubulin, and their relative distributions were determined by densitometry. Microtubule binding Microtubules were.