Ezrin

Paraffin sections of spheroids were deparaffinized and immersed in 10 mM citrate buffer (pH 6.0) (Wako) followed by autoclaving at 121 °C for 10 minutes. Sections were reacted with Primary antibody against zonula occludens-1 (ZO-1; Thermo Fisher) and stained with a Dako ENVISION Kit/HRP (DAB). Primary antibodies against Claudin-1 (Thermo Fisher), Ezrin (Santa Cruz), and Multidrug resistance-associated protein 2 (MRP2) (M2 III-6; Abcam) were incubated and stained with Alexa 488- or Alexa 568-conjugated goat anti-mouse or anti-rabbit IgG secondary antibodies and counterstained with Hoechst 33342 (Dojindo).

All cell lines were obtained from American Type Culture Collection. MCF10A cells were cultured as described (Debnath et al., 2003). MCF7 and HEK293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) plus 10% heat inactivated FBS. Cell Dissociation Buffer, enzyme-free, PBS-based (Life Technologies). Oligonucleotides of small interfering RNA (siRNA) for LPA receptors, G protein alpha subunits, and RhoGEFs were synthesized by QIAGEN. Oligonucleotides of siRNA for mDia1 were purchased from IBA GmbH. LPA (1-Oleoyl Lysophosphatidic Acid) and EDG family inhibitor Ki16425 were purchased from Cayman Chemical Company. Poly (2-hydroxyethyl methacrylate) (PolyHEMA) was purchased from Polysciences Inc. Antibodies against EDG4 were from Assay Biotechnology; LPAR receptors, Ezrin and LARG from Santa Cruz Biotechnology; PDZ-RhoGEF from IMGENEX; pMLC2 from Sigma and mDia1 from BD Biosciences. pCMV6-XL5 LPAR2 expression vector were purchased from OriGene (SC117226). pWPXL-based lentiviral expression vectors for H2B, LPAR2, Gα12, and Gα12Q/L were generated using standard PCR-based procedures or in the case of LifeAct-GFP were a kind gift from Oliver Fackler. Delipidized FCS was from Bio&SELL e.K.

After mice were sacrificed by CO₂ asphyxiation, eyes were enucleated and submerged in ice-cold 4% paraformaldehyde (PFA) in PBS or acetic acid/methanol/PBS (1:3:4) overnight. Standard IHC was conducted as described [39 (link)]. Antibodies against rhodopsin (RET-P1; Thermo Fisher Scientific, MS-1233-R7, dilute each drop to 250 μL), ezrin (Cell Signaling, 3145, rabbit polyclonal, 1:200), ezrin (Santa Cruz, sc-6409, 1:200), and cathepsin B (Cell Signaling, 31718T, rabbit monoclonal, 1:200) were used. These antibodies detected bands of expected or reported sizes on western blot analyses.

Total proteins were extracted by using RIPA with PMSF according to the manufacturer’s protocol. An equal amount (30 µg) of samples was separated on 10% SDS-PAGE gels and transferred to PVDF membranes (Millipore, Burlington, MA, USA). Then, the membranes were blocked in 5% non-fat milk for 1 h. Eventually, these membranes were detected using the ECL detection Kit (Solarbio, Beijing, China) after incubating the primary antibodies including anti-AJAP1 (AJAP1; Abcam; ab205496, rabbit secondary antibody) and anti-Ezrin (Ezrin; Santa Cruz Biotechnology, sc-58758, mouse secondary antibody).

Cell lysates were resolved in SDS-PAGE and transblotted onto polyvinylidene difluroide (PVDF) membrane. Primary antibodies used were phospho-Ezrin (T567), E-cadherin, Snail, β-catenin, vimentin, 14-3-3 ζ, Phopho-Akt (Ser-⁴⁷³) (1:1000Cell Signalling Technology), Ezrin, ERα (1:500, Santa Cruz), β-actin 1:2000 (Sigma Aldrich). The blots were incubated overnight with primary antibodies at 4 °C. HRP conjugated anti-mouse and anti-rabbit (1:10000, Sigma Aldrich) were used as secondary antibodies. Immunoreactive proteins were detected by enhanced chemiluminescence.

Complete protein extracts (30 µg) were loaded onto SDS-PAGE gels and electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes (Thermo Fisher Scientific). Then, the membranes were blocked with either 5% (w/v) non-fat dry milk or 5% (w/v) bovine serum albumin (BSA) (Sigma-Aldrich) in phosphate-buffered saline buffer containing 0.1% Tween-20 (Sigma-Aldrich) and incubated with primary antibody overnight at 4 °C. horseradish peroxidase (HRP)-conjugated secondary antibody incubation was performed at room temperature for 1 h. Protein immunodetection was performed using ECL™ Western Blotting Detection Reagents (GE Healthcare, Buckinghamshire, UK). The following primary antibodies were used: Cx43 (Sigma-Aldrich; #C6219; 1:8000), vinculin (Sigma-Aldrich; #V4505; 1:1000), ROCK1 (Santa Cruz Biotechnologies,; #sc-17794; 1:100), ezrin (Santa Cruz Biotechnologies; #sc-5875; 1:1000), vimentin (Santa Cruz Biotechnologies; #sc-6260; 1:1000), and RhoA (Cytoskeleton Inc., Denver, CO, USA; #ARH03; 1:1000). The secondary antibodies were rabbit anti-mouse horseradish peroxidase and goat anti-rabbit horseradish peroxidase (Life Technologies, Carlsbad, CA, USA; 1:10,000).