Sc 8426

Protein extract (50 μg) was separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and was electrophoretically transferred onto a polyvinylidene fluoride membrane (Millipore, Darmstadt, Germany). The membranes were blocked for 30 min at room temperature with 5% non-fat dried milk and incubated for 1 h with monoclonal antibodies against E-cadherin (sc-8426, mouse, IgG₁), N-cadherin (sc-7939, rabbit, IgG), γ-catenin (sc-30997, goat, IgG), insulin-like growth factor 1 receptor (IGF-1R; sc-sc462, mouse, IgG₁) or β-actin (sc-1616, goat, IgG) (Santa Cruz Biotechnology, Dallas, TX, USA). The antibodies against E-cadherin, N-cadherin and γ-catenin were at a dilution of 1:1,000 and the antibody against β-actin was at a dilution of 1:5,000. Subsequent to washing with PBS-T (10 mm Tris, pH 8.0; 150 mm NaCl; 0.5% Tween 20; Sheng Gong), the membranes were incubated for 1 h with secondary HRP-linked polyclonal goat anti-rabbit antibody (sc-2004, IgG, Santa Cruz Biotechnology), at a 1:20,000 dilution. The membranes were washed again with PBS-T and the proteins were visualized using ECL chemiluminescence and exposed to X-ray film.

The hCECs were cultured in a slide chamber and treated with ROCK inhibitors and incubated for 24 h. After removing the media, they were washed with phosphate-buffered saline (PBS), and fixed for 20 min in 3.7% (v/v) formaldehyde solution. Cells were permeabilized for 10 min with 0.5% (v/v) Triton X-100 and blocked for 1 h with 1% (w/v) bovine serum albumin (BSA) at room temperature. After washing with PBS, the cells were incubated overnight with either rabbit anti-Ki67 antibody (sc-23900, Santa Cruz, CA, USA), ZO-1 (sc-10804), β-catenin (ab32572), integrin β1 (sc-18887), SOX2 (sc-365823), N-cadherin (sc-59987), or E-cadherin (sc-8426) at 4 °C, and then washed with PBS. Cells are incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG antibody (1:100) or anti-mouse IgG antibody for 1 h at 37 °C in the dark. Cells were counterstained with Hoechst33342 nuclear staining dye. After extensive washing with PBS, the slides were mounted in a drop of mounting medium to reduce photobleaching. Negative control staining was conducted in parallel with the omission of the primary antibodies.

Protein was collected and extracted from A549 and H1299 cells with RIPA lysis buffer and protease inhibitor cocktail and protein phosphatase inhibitor. The samples were then transferred to PVDF membranes, following electrophoresis and the membranes were incubated with rabbit-anti SMAD3 (Cell Signaling Technology, Inc.) or mouse-anti E-cadherin or mouse-anti N-cadherin (both Santa Cruz Biotechnology, Inc.) primary antibodies(SC-8426 for E-cadherin, SC-8424 for N-cadherin) overnight at 4 °C. The following day, the membranes were incubated with indicated secondary antibodies (Santa Cruz Biotechnology, Inc, SC-2005) for 1 h at room temperature. Detection was performed using the electrochemiluminescence kit (Pierce; Thermo Fisher Scientific, Inc.). β-actin (Santa Cruz Biotechnology, Inc.) was used as the internal control.

Equal amounts of cell lysates were electrophoretically separated and transferred to a PVDF membrane. After blocking with 5% skimmed milk, the membrane was incubated with primary antibodies against SOX9 (AB5535, 1:2000; Millipore), E-cadherin (sc-8426, 1:100; Santa Cruz), vimentin (sc-6260, 1:100; Santa Cruz), β-actin (sc-7963, 1:100; Santa Cruz), and appropriate secondary antibodies. The signals were detected using enhanced chemiluminescence (GE Healthcare).

The mESCs colonies were fixed with 4% PFA for 20 min at room temperature, washed three times with PBS and permeabilized with 0.1% Triton X-100 at room temperature for 20 min. After three washes with PBS, the cells were blocked with 5% FBS for one hour at room temperature and incubated with diluted primary antibody overnight. The next day, the cells were washed three times with PBS and incubated with fluorophore-conjugated secondary antibody for one hour at room temperature and followed by DAPI staining. The images were taken under the fluorescence microscope (EVOS FL, Thermo Scientific). The following antibodies were employed in this study: anti-SSEA1 (1:100, 4744s, CST), anti-Nanog (1:300, ab80892, Abcam), anti-Oct-3/4 (1:200, sc-5279, Santa Cruz), anti-E-cadherin/CDH1 (1:1000, sc-8426, Santa Cruz), anti-Neuronal Class III β-Tubulin (1:1000, MMS-435P, Covance), anti-DESMIN (1:500, MAB3430, Millipore), anti-AFP (1:50, MAB1368, R&D System).