Anti zo 1

For protein extraction, cells were washed with ice-cold PBS and lysed for 20 min on ice with RIPA (RadioImmunoPrecipitationAssay) buffer (150 mM NaCl, 50 mM Tris pH7.4, 1 % NP40, 1 % sodium-deoxycholat, 1 mM EDTA, 1 mM Na₃VO₄, 25 mM NaF, 1 mM PMSF, 5 mM beta-glycerolphosphat, protease inhibitor cocktail tablets (Complete mini; Roche)). Total cell lysates were cleared by centriguation (10 min, 4 °C, 10,000 g). Protein concentration was measured with the Bradford ProteinAssay (BioRad). Western blots were performed as described in [50 (link)] with the following antibodies: anti-Fibronectin: Santa Cruz, sc-9068; anti-ZO1: Zymed Laboratories, 33–9100; anti-E-Cadherin: BD Transduction Laboratories, 610181; anti-Vimentin: Sigma, V2258; anti-Actin: Sigma, A2066; anti-phospho-AKT (Ser473): Cell Signaling, 9271; anti-total-AKT: Cell Signaling, 9272; anti-phospho-ERK1/2: Sigma, M8159; anti-total-ERK1/2:Sigma,M5670; anti-Pan-Ras: Calbiochem, OP38.
For immunoprecipitation, cells were lysed as described for Western blotting. Equal amounts of protein were incubated with anti-v-H-Ras antibody (Calbiochem, OP01) overnight at 4 °C. Then ProteinA/G plus beads (Santa Cruz) were added and samples were incubated for 1 h at 4 °C. Thereafter, immune complexes were collected by centrifugation, washed twice with ice cold RIPA buffer and subjected to SDS PAGE and Western blotting.

After 24 hours, the ARPE-19 cells were seeded into 8-well cell chamber slides and transfected with miRNA (mimic/inhibitor). Next, they were incubated for up to 24 hours in the presence or absence of TGF-β2. After the cells were washed three times with PBS, they were fixed with 4% paraformaldehyde and then treated with 0.1% Triton X-100 for 10 min on ice. They were then incubated with 5% bovine serum albumin in PBS for 1 hour at room temperature. Anti-ZO-1 (1:200dilution; Zymed Laboratories) and antivimentin antibodies (1:200 dilution; Santa Cruz Biotechnology) were used as the primary antibodies. The secondary antibodies comprised DyLight 488 antirabbit immunoglobulin G (IgG) and DyLight 594 antimouse IgG antibodies (1:200 dilution; Bethyl Laboratories, Montgomery, TX, USA), respectively. The nuclei were counterstained with 4’,6’-diamidino-2-phenylindole (Sigma-Aldrich). The preparation was fixed with 70% glycerol and examined using a fluorescence microscope (CKX41, Olympus Corporation, Tokyo, Japan).

The small intestines from mice were divided into approximately 1-cm portions, and each portion was opened with a longitudinal cut. After several washes with PBS, intestines were fixed with 4% PFA for 2 h and then perfused with 10% sucrose. Intestinal sections were embedded in optimal cutting temperature (OCT) compound and frozen to −80°C. A cryostat was used to cut 5- to 7-μm-thick transverse intestine slices, which were mounted onto microscope slides. The slices were washed with PBS, incubated with 2 N HCl for 30 min, and washed three times with 0.1 M Na₂B₄O₇ (pH 9). For BrdU and ZO-1 labeling, the slices were permeabilized with PBS–0.1% Triton X-100–10% horse serum for 30 min and then incubated with anti-BrdU (Abcam; Ab 1893) and anti-ZO-1 (Zymed Laboratories 33-9100), followed by anti-sheep Alexa Fluor 637 and anti-mouse Alexa Fluor 488 secondary antibodies (Invitrogen), respectively, or phalloidin (Invitrogen). Immunohistochemistry images were acquired in a blind manner for BrdU, as images were selected by DAPI (4′,6-diamidino-2-phenylindole) and ZO-1 staining. Samples were all imaged using a confocal laser scanning microscope (LSM510 or LSM710; Zeiss).