Sc 59987

After 24 h, transfected cells were washed once with ice-cold PBS 1× and after dislodged by scraping using Buffer NP40 (50 mM Tris-HCl pH7,5; 150 mM NaCl; 1% Nonidet P 40 substitute). Cells were lysed for 30 min at 4 °C on a rotator and the lysates were cleared by centrifugation (17,949 × g for 30 min at 4 °C).
Plasma membrane proteins were extracted using the Plasma Membrane Protein Extraction Kit from Abcam. Proteins from membrane and cytoplasmic extracts were western blotted with antibodies against PKP2 (EB10841, Everest Biotech, 1:1000), N-cadherin (sc-59987, Santa Cruz Biotechnology, 1:1000), and GAPDH (sc-32233, Santa Cruz Biotechnology, 1:1000). Secondary antibodies were anti-goat (ABIN2169607, Antibodies online, 1:4000) and anti-mouse (ABIN6699027, Antibodies online, 1:4000) as appropriate. Immunoblots were developed in an iBright 1500 system.

An antibody against NNMT (sc‐376048; Santa Cruz Biotechnology) was used for immunoblotting (1:100) and immunohistochemistry (1:100). Antibodies for immunoblotting against ERK1/2 (1:1000; no. 4695), phospho‐ERK1/2 (Thr202/Tyr204) (1:2000; no. 4370), H3K4me3 (1:1000; no. 9751), and H3K27me3 (1:1000; no. 9733) were purchased from Cell Signaling Technology. Antibodies against neural cadherin (N‐cadherin) (1:200; sc‐59987; Santa Cruz Biotechnology) and H3K9me2 (1:500; ab1220; Abcam) were used for immunoblotting. An antibody against Cleaved Caspase‐3 (no. 9661; Cell Signaling Technology) was used for immunohistochemical analyses (1:400). Antibodies against β‐actin (1:1000; no. 5125) and Histone H3 (1:1,000; no. 4499), used as loading controls for immunoblotting, were obtained from Cell Signaling Technology.

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.