MICAL-L2 was displaced from TJs upon actin depolymerization and was distributed along radiating actin cables and stress fibers in Ca2+-depleted MTD-1A and fibroblastic NIH3T3 cells, respectively

MICAL-L2 was displaced from TJs upon actin depolymerization and was distributed along radiating actin cables and stress fibers in Ca2+-depleted MTD-1A and fibroblastic NIH3T3 cells, respectively. was distributed along radiating actin cables and stress fibers in Ca2+-depleted MTD-1A and fibroblastic NIH3T3 cells, respectively. These results suggest that MICAL-L2 mediates the endocytic recycling of occludin and the formation of functional TJs by linking Rab13 to actin cytoskeleton. We rename MICAL-L2 as JRAB (strain DH5 and purified using glutathione-Sepharose beads (GE Healthcare, Piscataway, NJ) according to the manufacturer’s instructions. Two milligrams of GST or GST-JRAB-C protein was immobilized on HiTrap NHS-activated columns (GE Healthcare) according to the manufacturer’s instructions. Two female Wistar rats were immunized with 100 g of GST-JRAB-C protein twice at 4-wk intervals, after which whole blood was collected. Crude immunoglobulin (Ig) fractions were D-Luciferin prepared by ammonium sulfate precipitation and exceeded through a GST-immobilized column to remove an anti-GST antibody. The anti-JRAB polyclonal antibody was further purified on a GST-JRAB-C-immobilized column according to the manufacturer’s instructions. The rat anti-occludin (MOC37) antibody was a kind gift from Dr. S. Tsukita (Kyoto University, Kyoto, Japan). Rabbit anti-ZO-1 and mouse anti-TfR antibodies were purchased from Zymed Laboratories (South San Francisco, CA), mouse anti-Myc (9E10) was from American Type Culture Collection (Manassas, VA), mouse anti-HA (12CA5) and rat anti-HA (3F10) were from Roche Diagnostics (Mannheim, Germany), and rabbit anti-green fluorescent protein (GFP) was from Invitrogen (Carlsbad, CA). Cell Culture and Transfection MTD-1A and L cells were kindly supplied by Dr. S. Tsukita. Baby hamster kidney (BHK), MDCK, NIH3T3, and Caco2 cells were obtained from American Type Culture Collection. BHK, MTD-1A, and NIH3T3 cells were cultured at 37C (5% CO2 and 95% air) in DMEM with 10% fetal bovine serum (FBS). BHK and MTD-1A cells were transfected using Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer’s instructions. Coimmunoprecipitation Thirty-six hours after transfection, BHK and MTD-1A cells were lysed with 25 mM Tris-HCl, pH 7.5, containing 0.5% 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate (CHAPS), 125 mM NaCl, 1 mM MgCl2, 10 g/ml (4-amidinophenyl)-methanesulfonyl fluoride (APMSF), and 100 M guanosine 5-for 10 min at 4C, and the resulting postnuclear supernatant (PNS) fractions were taken. The PNS fractions were further centrifuged at 100,000 for 60 min at 4C in a Hitachi S100AT2 rotor (Hitachi, Tokyo, Japan) to obtain S100 and P100 fractions. PNS, S100, and P100 fractions were subjected to Western blot analysis. Western Blot Each sample was separated by SDS-PAGE, and proteins were transferred to a polyvinylidene diflouride (PVDF) membrane. Membrane blocking and antibody dilutions were done in Block Ace (Dainippon Pharmaceutical, Osaka, Il1b Japan). Blots were developed by chemiluminescence using a horseradish peroxidase-coupled secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) with an ECL-Plus kit (GE Healthcare). Quantitation was performed on scans of autoradiograph films with nonsaturated signals using NIH Image D-Luciferin 1.62 software (http://rsb.info.nih.gov/nih-image/). Immunofluorescence Microscopy BHK cells were grown on glass coverslips and fixed with 2% formaldehyde in phosphate-buffered saline (PBS) for 15 min at room temperature. MTD-1A and NIH3T3 cells, produced on Transwell filters (Corning, Acton, MA) or glass D-Luciferin coverslips, were fixed with 10% trichloroacetic acid in PBS for 15 min on ice or 1% formaldehyde in PBS for 15 min at room heat. After permeabilization with 0.2% Triton X-100 in PBS for 15 min and blocking with D-Luciferin 5% goat serum in PBS for 60 min at room temperature, cells were incubated with primary antibodies for 60 min and with Alexa 488- or Alexa 594-conjugated secondary antibodies (Invitrogen) for 60 min at room heat. F-actin was labeled with rhodamine-phalloidin (Invitrogen). Fluorescent images were acquired using a Radiance 2000 confocal laser scanning microscope (Bio-Rad, Hercules, CA). Recombinant Adenovirus Contamination Recombinant adenovirus expressing EGFP.