Statistical significance was determined with GraphPad Prism by unpaired Student’s test or analysis of variance test as indicated (*, 0

Statistical significance was determined with GraphPad Prism by unpaired Student’s test or analysis of variance test as indicated (*, 0.05; **, 0.01; ***, 0.001). Results Elevated AnxA6 Levels Reduce Cell Migration and Invasion The reduced FN secretion in cells with high levels of AnxA6 (15) prompted us to examine cell migration. that elevated AnxA6 levels cause LE cholesterol accumulation, we propose that AnxA6 and blockage TAK-242 S enantiomer of LE cholesterol transport are critical for endosomal function required for Stx6-mediated recycling of integrins in cell migration. for 10 min at 4 C. Proteins from supernatants (500C800 g) were incubated with 2 g of mouse monoclonal anti-Stx6, rabbit polyclonal anti-VAMP4, or mouse/rabbit IgG for 2 h at 4 C, respectively, followed by an additional 60-min of incubation upon addition of TAK-242 S enantiomer protein G-Sepharose. Immunoprecipitates were washed twice in TGH made up of 150 mm NaCl and once in TGH without NaCl and analyzed for Stx6, VAMP3, Stx16, VAMP4, and Vti1a (15). Microscopic Techniques and Image Analysis Cells were produced on coverslips, fixed with 4% paraformaldehyde for 20 min, washed, permeabilized with 0.1% saponin for 10 min, blocked with 1% BSA for 5 min, and incubated with primary and secondary antibodies. Alternatively, cells were permeabilized with 0.1% Triton X-100 for 5 min. In some experiments, cells were seeded onto FN-coated coverslips; therefore coverslips were washed twice with PBS, coated with poly-l-lysine (50 g/ml) in PBS for 2 h, washed twice with PBS, incubated in 20 g/ml FN for 3 h, and washed twice with PBS before use. Finally, samples were mounted in Mowiol, and cells were observed using a TAK-242 S enantiomer Leica DMI 6000B epifluorescence inverted microscope equipped with an HCX PLA Apo 63 oil immersion objective. Some images were captured with a Leica TCS SP5 laser scanning confocal microscope equipped with a DMI6000 inverted microscope, blue diode (405 nm), argon (458/476/488/496/514 nm), diode-pumped solid state (561 nm), HeNe (594/633 nm) lasers, and Apo 63 oil immersion objective lenses. Image analysis was performed with NIH ImageJ software (26). Co-localization analysis was done using the ICA (intensity correlation analysis) plug-in. To quantify staining intensity, images were captured using identical CFD1 microscope settings. Isolation of Subcellular Fractions Subcellular fractionation of CHO-WT and CHO-A6 membranes on discontinuous sucrose gradients was performed, and the distribution of Stx6, RE (VAMP3), for 20 min at 4 C. Equal amounts of protein from the supernatant were incubated for 1 h with streptavidin beads to precipitate biotinylated proteins, which were analyzed by immunoblotting. Integrin recycling was measured as described previously (28). In brief, cell surface biotin-labeled cells were incubated for an additional 30 min to allow internalization of surface biotinylated proteins (quadruplicates for each cell line). One plate was lysed, whereas the three other plates were washed twice in HBSS followed by two washes in PBS, 0.5 mm EDTA. The remaining surface biotin was TAK-242 S enantiomer removed by incubating cells with reduced l-glutathione buffer (50 mm reduced l-glutathione, 75 mm NaCl, 2 mm EDTA, 75 mm NaOH, 0.1% BSA). Reduced l-glutathione was neutralized with 10 mm iodoacetamide in HBSS. Cells from a second plate were then lysed, and the remaining two plates were incubated for 30 min in complete cell culture medium. One plate was lysed, whereas the other plate was incubated with reduced l-glutathione and iodoacetamide as described above to remove the surface biotin from recycled proteins. Multiscratch Assays Multiscratch signaling assays were performed as described (29). In brief, 5 105 cells were seeded onto 6-well plates and produced to 90% confluence. Using a 200-l pipette tip, five vertical and five horizontal scratches were made, TAK-242 S enantiomer and lysates were prepared at 0, 30, and 60 min postscratch. Cell lysates were analyzed by Western blotting for total and phosphorylated (Tyr(P)861) focal adhesion kinase (FAK) and (Tyr(P)527) Src. CTxB and STxB Uptake Cells were incubated in DMEM, 10% FCS with fluorescently labeled CTxB and STxB (CTxB-Cy5, 2 g/ml; STxB-Cy3, 1 g/ml) for 10 min at.