(2007) A dileucine motif in its cytoplasmic domain directs -catenin-uncoupled E-cadherin to the lysosome. lacks dileucine residues, exhibited partial apical-lateral polarity that JMS-17-2 was abolished by mutation of its ankyrin-binding site but was not affected by clathrin knockdown. The polarity motif thus integrates complementary activities of lateral membrane retention through ankyrin-G JMS-17-2 and apical-lateral transcytosis of mis-localized protein through clathrin. Together, the combination of retention and editing function to ensure a high fidelity steady state localization of E-cadherin at the lateral membrane. for 4 h. MDCK cells were infected with lentivirus in the presence of 8 g/ml Polybrene overnight. After 48 h, cells were sorted by mCherry fluorescence using fluorescence-activated cell sorting (FACS). For lateral membrane biogenesis, stable cells lines were preinduced at confluence for 48 h with 5 g/ml doxycycline, then trypsinized and plated at confluence in 14-mm insert MatTek plates. Cells were fixed at the indicated times and processed for immunocytochemistry. Parallel samples were also prepared for Western blot analysis. Immunoblots Samples (10-l volume) were run on a 3.5C17.5% gradient gel in 1 Tris buffer, pH 7.4 (40 mm Tris, 20 mm NaOAc, and 2 mm NaEDTA) with 0.2% SDS (49). Transfer to nitrocellulose was performed overnight at 300 mA at 4 C in 0.5 Tris buffer with 0.01% SDS. Membranes were blocked with Blot buffer I (150 mm NaCl, 1 mm NaN3, 1 mm EDTA, 0.2% Triton X-100, and 10 mm phosphate buffer, pH 7.4) with 2% bovine serum albumin and incubated overnight at 4 C with primary antibodies diluted in blocking buffer. For experiments involving mouse primary antibodies, membranes were washed with blot buffer I and incubated in rabbit anti-mouse IgG for Rabbit polyclonal to AGAP 1 h at room temperature. Membranes were then incubated with I125Clabeled protein A/G (1:1000). Membranes were placed on a storage phosphor screen, and signal was detected using a Typhoon imager (GE Healthcare). Apical Mislocalization MDCK cells grown on MatTek plates were transfected with 50 ng of cDNA encoding V5-E-cadherin-GFP or mutants using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. After 5 h of transfection, cells were fed, and doxycycline (or vehicle control for uninduced samples) was added to the medium at a concentration of 5 g/ml. 48 h after transfection cells were fixed and prepared for immunocytochemistry as described above. Confocal stacks of MDCKII cells were captured using a Zeiss LSM 780 using a 100 1.45 PlanFluor oil objective with 0.25-micron Z spacing and pinhole set to 1 Airy unit. A three-dimensional region corresponding to the apical surface or lateral JMS-17-2 membrane was drawn using Volocity software (PerkinElmer Life Sciences), and mean pixel intensity was quantified and expressed as mean pixel intensity on the apical membrane mean pixel intensity on the lateral membrane. Apical E-cadherin Editing To monitor the fate of apically localized E-cadherin, MDCK cells grown on Transwell filters (Corning) were transfected with 50 ng of cDNA encoding V5-E-cadherin-GFP using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. 48 h after transfection, cells were chilled on ice to minimize protein trafficking and incubated on the apical surface with mouse anti-V5 antibodies in DMEM for JMS-17-2 1 h on ice to mark apically localized protein. Cells were quickly washed in cold DMEM and warmed to 37.