Rabbit anti β catenin

Enucleated eyes were fixed in 4% formaldehyde in 1x PBS for overnight at 4 °C, washed 3 × 10 minutes in 1x PBS and followed by paraffin embedding. For histology analysis, paraffin sections (6 µm) were deparaffinised, rehydrated with ethanol series and stained with Hematoxylin and Eosin (H & E). For immunofluorescence, paraffin sections (6 µm) were deparaffinised, rehydrated with ethanol series, followed by antigen retrieval in sodium citrate buffer (10 mM sodium citrate buffer, 0.05% Tween 20, pH-6.0). The sections were blocked in 10% BSA/0.1% PBT for 30 minutes and incubated with primary antibodies in 1% BSA/0.1% PBT for overnight. On the following day, samples were washed in 1x PBS for 3 × 10 minutes, incubated with secondary antibodies in 1% BSA/0.1% PBT for 1 hour, washed in 1x PBS, counterstained with DAPI (1 µg/ml) and mounted in Mowiol (Sigma). Following are the antibodies used for this study: rabbit anti-β-catenin (1:1000, Sigma, C2206), rabbit anti- Pax6 (1:1000, Covance, PRP-278P), goat anti-K12 (1: 300, Santa Cruz Biotec Inc, Sc-17101), anti-rabbit Alexa 594, and anti-goat Alexa 594 (all 1:500, Molecular Probes). Stained sections were analysed with Zeis Imager Z2 (Zeis, Germany).

The primary antibodies used were as follows: mouse anti-β-catenin (BD Biosciences, 610153), rabbit anti-β-catenin (Sigma-Aldrich, C2206), rabbit anti-C11orf95/ZFTA (Abcam, ab170283), rat anti-CD24 conjugated with FITC (Thermo Fisher, 11-0242-82), rabbit anti-Doublecorin (DCX, cell signalling, #4604), mouse anti-FoxJ1 (eBiosciences, 14-9965), mouse anti-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Millipore, MAB374), chicken anti-GFAP (Aves, GFAP), rabbit anti-RFX1 (Novus, NBP1-52654), rabbit anti-S100β (Proteintech, 15146-1-AP), rabbit anti-smooth muscle actin (αSMA, Proteintech, 55135-1-AP), mouse anti-acetylated tubulin (Sigma-Aldrich, T6793) and rabbit anti-γ-tubulin (Sigma-Aldrich, T5192). The secondary antibodies used were as follows: conjugated to Alexa Fluor dyes (Abcam) or HRP (Jackson ImmunoResearch).

MDCK cells were grown on coverslips during cell seeding. At the end of the corresponding treatments cells were washed with PBS and fixed with 4% (m/v) paraformaldehyde at 25° C for 20 min, and permeabilized with 0.1% (v/v) Triton X-100 at 25° C for 20 min. After washing with PBS, cells were incubated with 3% BSA in PBS at 25° C for 60 min, followed by incubation for 90 min with the primary antibodies: 1:250 mouse anti-α-catenin (polyclonal, C2081; Sigma), 1:200 rabbit anti-β-catenin (monoclonal, C7082; Sigma), 1:50 rabbit anti-E-cadherin (polyclonal, SC-7870, Cruz Biotechnology), 1:300 mouse anti-giantin (monoclonal, ab37266; Abcam), 1:50 rabbit anti-SNAI1 (polyclonal, SC-281999; Santa Cruz Biotechnology) and 1:50 rabbit anti-SNAI2 (polyclonal, SC-15391; Santa Cruz Biotechnology). Primary interactions were detected by using a 1:200 F(ab ´)₂ fragment of goat anti-rabbit IgG and a 1:200 F(ab ´)₂ fragment of goat anti-mouse IgG (polyclonal, 115-095-166, 115-024-166, 111-025-144, 111-095-003; Jackson ImmunoResearch), both FITC or TRITC. Actin filaments were visualized by 1 μg/mL phalloidin-FITC (P5282, Sigma-Aldrich) and the ER with 0.20 μg/mL concanavalin A-TRITC (C860; Thermo Fisher Scientific). The coverslips were then mounted onto microscope glass slides with Vectashield Mounting Media (H-1000; Vector Laboratories) and stored at -20°C until analysis.

The following primary antibodies were used in this study: mouse anti-MyHC (Santa Cruz, Heidelberg, Germany), mouse anti-MyoG (Sigma), rabbit anti-Atrogin-1 (Novus Biological, Abingdon, UK), rabbit anti-Ubiquitin (Sigma), rabbit anti-LC3B (Sigma), rabbit anti-MyoF ((Novus Biological), rabbit anti-Axin-1 (Sigma), rabbit anti-GSK3β (Abcam, Cambridge, UK), rabbit anti-β-catenin (Sigma), rabbit anti-Dvl-2 (Abcam), rabbit anti-p62 (Sigma), rabbit anti-LC3B (Sigma), rabbit anti-GAPDH (Santa), and rabbit anti-Histone H3 (Abcam). The following secondary antibodies were used: mouse anti-rabbit (Santa Cruz), goat anti-rabbit (Santa Cruz), mouse anti-rabbit horseradish peroxidase, goat anti-mouse HRP, and donkey anti-goat HRP (Sigma). Myotube atrophy was induced by treatment with 10 μM dexamethasone (Sigma) for 24 h.

Mouse organs were rinsed with PBS, homogenized with a glass homogenizer in ice-cold lysis buffer (40 mM Tris-HCl, pH 7.6, 1% Triton X-100, 40 mM β-glycero-Phosphate, 100 mM NaCl, 1 mM EDTA, 5% glycerol, 50 mM NaF, 0.1 mM sodium orthovanadate, 0.1 mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 0.7 μg/ml pepstatin), and processed for SDS-PAGE and Western blotting as previously described63 (link). Primary antibodies used were: mouse anti-E-cadherin (1/4000, Invitrogen), rabbit anti-β-catenin (1/4000, Sigma), mouse anti-Actin (1/10000, Millipore), rabbit anti-aPKC (1/1000, Santa Cruz Biotechnology), rabbit anti-Par3 (1/1000, Millipore) and rabbit anti-PALS1 (1/1000, Santa Cruz Biotechnology). HRP-conjugated secondary antibodies were from GE Healthcare and were used at a 1/1000 dilution.