At least two microvilli were measured per cell and a total of 300 microvilli were measured from each of the control and KD groups. Immunoelectron microscopy Ultrastructural localization studies were carried out on control and myosin VI KD Caco-2/Bbe cells grown on polycarbonate filters (Transwell, 0.4?m, Corning Inc.) for 12 days then infected with Ad-HA-NHE3. that myosin VI also moves NHE3 down the microvillus. (Hegan, et al., 2012; T.C. and M.D., unpublished observations). Finally, the localization of the tight junction proteins ZO-1 (also known as TJP1) (supplementary material Fig. S1a,b), occludin (supplementary material Fig. S1c,d) and claudin-1 (supplementary material Fig. S1e,f) were similar in control and KD cells, as were the trans-epithelial resistances (Fig.?2E). Open in a separate window Fig. 2. Efficient KD of myosin VI in Caco-2/Bbe cells did not significantly alter the ultrastructure of their microvilli. (A) Myosin VI expression in lenti-shRNA virus-infected Caco-2/Bbe cells. Caco-2/Bbe cells were infected with empty vector (lane 1), lentiviral vector encoding shRNA against GFP (lanes 2 and 3), and PD173074 three different shRNAs against myosin VI (shMyoVI) [no. 1 (lane 4), no. 2 (lane 5) and no. 3 (lane 6) (sequences are shown in supplementary material Table S1)]. After 48?h of incubation, the cells were harvested for western blot analysis. This experiment was repeated three times with similar results. (B,C) TEM of microvillus in control (B) and myosin VI KD (C) cells grown on Transwell filters for 14 days. Representative images are shown. No obvious morphological changes were seen in the myosin VI KD cells, such as PD173074 pulling away of microvillar membrane from the base of microvillus, as previously reported in myosin VI KO mouse intestine (Ameen and Apodaca, 2007). Scale bar: 500 nm. (D) The lengths of microvilli in myosin VI KD cells were not significantly KLF4 (for 10?min and the post-nuclear supernatant was collected, protein concentrations were measured by a Bradford assay [Sigma-Aldrich (St. Louis, MO)] and adjusted to 1 1?g/l. Of the 1?ml of cell lysate supernatant, 0.9?ml was incubated with streptavidinCAgarose beads (Pierce Chemical, Rockford, IL) for 3?h at 4C. After sedimenting the beads, the supernatant was retained as the intracellular fraction and the avidinCagarose beads were washed five times in N? buffer (60?mM HEPES pH?7.4, 150?mM NaCl, 3?mM KCl, 5?mM Na3EDTA and 3?mM PD173074 EGTA) with 0.1% Triton X-100 to remove nonspecifically bound proteins. The proteins bound to the avidinCagarose beads, which represent plasma membrane NHE3, were solubilized PD173074 in 90?l of loading buffer (5?mM Tris-HCl pH?6.8, 1% SDS, 10% glycerol and 1% 2-mercaptoethanol), boiled for 10?min. Two dilutions (30?l and 60?l) of total lysate, surface and intracellular proteins from each group were loaded, size-fractionated by SDS-PAGE (10% gel) and then electrophoretically transferred onto nitrocellulose. After blocking with 5% nonfat milk in PBS, the blots were probed with monoclonal anti-HA antibody, rinsed, incubated with anti-mouse-IgG conjugated to IRDye? 488 secondary antibodies (LI-COR) and visualized. Signals were quantified on an Odyssey Infrared Imaging System (Li-Cor, Lincoln, NE). The signal intensity derived by linear regression was used to obtain a single value for each sample. The percentage of surface NHE3 was calculated [(surface NHE3 signal/total NHE3 signal) dilution factor PD173074 of surface and total NHE3 samples] and expressed as percentage of total NHE3. Immunocytochemistry, confocal microscopy and image quantification Caco-2/Bbe cells were grown on Anapore filters (25?mm, 0.02?m, Nunc) then infected at 12 days post-confluence with Ad-HA-NHE3 as described above. After 48?h, cells were washed with ice-cold PBS and fixed for 30?min at 4C with 3% paraformaldehyde (PFA) in PBS. For dynasore treatment, cells were serum starved for 4?h and incubated with dynasore (80?M for 30?min) or an equal volume of DMSO and then processed for fixation. Fixed cells were blocked and permeabilized with 1% BSA and 0.075% saponin in PBS for 1?h at 4C. Cells were incubated 1?h at room temperature with primary antibodies in 1% BSA in PBS, rinsed in 0.1% BSA in PBS (three times for 5?min), then incubated with secondary antibodies in 1% BSA in PBS for 1?h, and rinsed again in 0.1% BSA in PBS (three times for 5?min). Cells were rinsed with 0.1% BSA and 0.075% saponin in PBS prior to mounting. Membrane inserts were detached from wells, placed on glass microscope slides, mounted with Fluorogel (Invitrogen), and examined with a Zeiss LSM510 confocal.