Ecl blocking agent

Cells were lysed in lysis buffer (1 % NP-40, 20 mM HEPES, 150 mM NaCl, 10 % glycerol, 2 mM Na₃VO₄, 10 mM Na₄P₂O₇, 2 mM NaF and Complete EDTA-free Protease Inhibitor Cocktail (Roche, Mannheim, Germany)). Total lysate (10 μg) was loaded on an 11 % acrylamide gel and subjected to SDS-PAGE. Proteins were transferred to PVDF membrane overnight. Membrane was incubated for 1 h in blocking buffer (Tris-buffered saline containing 0.1 % Tween20 and 2.5 % ECL Blocking Agent (GE Healthcare, Little Chalfont, UK)) and for 2 h at RT with mouse monoclonal anti-PTTG1 antibody (DCS-280; Abcam; Cambridge, MA) diluted 1:1000 in blocking buffer, followed by alkaline phosphate-conjugated anti-mouse IgG (Millipore, Billerica, MA) diluted 1:4000 in blocking buffer for 1 h at RT. Proteins were visualised using ECL detection reagent (GE Healthcare) on a Typhoon FLA 7000 IP² (GE Healthcare).

Levels of mBDNF, TrkB-FL, and TrkB-T1 were detected in the retina and SC samples using western blotting. Equal amounts of the sample (retina: BDNF 120 μg, TrkB 50 μg; SC: BDNF 60 μg, TrkB 20 μg) were loaded onto 4–12% Bis-Tris polyacrylamide gels (BioRad Laboratories, CA, USA) and transferred to polyvinylidene difluoride (PVDF) membranes (BioRad Laboratories, CA, USA). The membrane was incubated for 2 h in 5% ECL Blocking Agent (GE Healthcare, UK) in 0.01 M tris buffered saline (TBS). Overnight incubation with primary antibody, polyclonal rabbit anti-BDNF (1:500, sc-546, Santa Cruz Biotechnology, TX, USA) or polyclonal rabbit anti-TrkB (1:1500, 07–225, Millipore, MA, USA), was followed by 45 min incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:10,000, Dako, Denmark). Specific protein bands were visualized using the luminol-based enhanced chemiluminescence kit (SuperSignal West Dura, Thermo Scientific, MA, USA). In order to normalize detected amounts of mBDNF or TrkB to the total amount of protein loaded, LavaPurple (Gelcompany, CA, USA) total protein stain was performed prior to primary antibody incubation. Specific protein bands and LavaPurple staining were imaged with the ChemiDoc MP Imaging System (BioRad Laboratories, CA, USA) and optical density analyses were performed using Image Lab 4.1 software (BioRad Laboratories, CA, USA).

Proteins expressions of E-cadherin (epithelial marker) and vimentin (mesenchymal marker) of OCUP-A1 and OCUP-A2 were examined by Western blotting. Approximately 30 μg of protein extracts were separated through 10 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride membrane using the Trans- Blot® Turbo™ Transfer System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Then, using the SNAP i.d.® 2.0 system (Merck Millipore, Darmstadt, Germany), the membranes underwent application of Tris-buffered saline-Tween (TBS-T) solution containing each primary antibody against anti-rabbit E-cadherin (1:300, Cell Signaling Technology, Danvers, MA, USA), anti-rabbit vimentin (1:300, Cell Signaling Technology), and anti-mouse β-actin (1:2500, Sigma-Aldrich, St. Louis, MO, USA) for 10 min after blocking with ECL blocking agent (GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK) and incubated with HRP-conjugated anti-mouse or anti-rabbit secondary antibody (1:5000, Sigma-Aldrich) for 10 min. Protein bands were visualized with the Luminescent Image Analyzer LAS 4000-plus (Fuji, Tokyo, Japan).

The region corresponding to target RNAs selected according to the RIPseq experiments was amplified from bacterial DNA adding a T7 promoter at the 5' end. PCR products were used in MEGAshortscript T7 Kit (Ambion) to produce in vitro RNA (S6 Table). 2μM of Biotin-11-CTP was added into the reaction mix for later detection. This reaction mix was incubated at 37°C for 2h and the RNA was purified by Phenol/Chloroform extraction. The RNA concentration was estimated by UV absorption at 260nm. For 10μl interaction assays, 200nM of RNA was combined with varying concentrations of purified CsrA-His (0–5μM) and incubated in buffer A in presence of 250ng tRNA yeast (Invitrogen) for 30min at RT. Subsequently, samples were fractionated under non-denaturing conditions on Blue-Native PAGE and blotted to BrightStar-Plus transfer membranes (Ambion). Membranes were blocked in PBS buffer containing 0.1% Tween-20 and 1% ECL blocking agent (GE Healthcare) for 1h at RT and, afterwards, incubated for 1h in the same buffer including mouse anti-biotin antibodies (Invitrogen). After washing and binding of secondary antibodies (anti-mouse Ig-HRP, Dako), the RNA-bands were visualized with ECL Plus Western blotting solutions (GE Healthcare) and detected with a G-box (Syngene).

RPE-J cells were cultured in OGD conditions by the same method described above. After treating cells with the indicated concentrations of CLT and exposing them to OGD conditions for 48 h, cells were washed with PBS, centrifuged, and then lysed using a ProteoJET Cell Lysis kit. Protein concentrations were determined using a bicinchoninic acid protein assay kit (Wako). Electrophoresis was performed using 4–15 % Tris–glycine gels. Proteins were transferred to PVDF membranes using a semidry transferring system (Biorad). The membranes were blocked with 5 % ECL blocking agent (GE Healthcare), incubated with primary antibodies against cleaved-caspase-3 (1:1000; Cell Signaling) and subsequently with the secondary antibody, horseradish peroxidase-linked IgG (1:10,000; Cell Signaling). After stripping the membranes of the antibodies for 10 min using reagents from a Western Re-Probe kit (Jacksun Biotech), the membrane was probed in a similar manner for β-tubulin (1:2000; Cell Signaling). Bands were visualized using an enhanced chemiluminescence system (ECL Plus, GE Healthcare). Band intensities were measured using Image J software.

After 48 hr of incubation, the cells were lysed in 20 mM HEPES (pH 7.4) containing 0.1% Triton X-100 and 1× complete protease inhibitor cocktail, 25x (Roche Diagnostics, Basel, Switzerland). Equal amounts of protein were separated by SDS–PAGE and transferred to a Hybond-LFP 0.2 polyvinylidene difluoride membrane (GE Healthcare, Buckinghamshire, UK). The membrane was blocked in ECL Blocking Agent (GE Healthcare) and subjected to immunoblotting with antibodies against E-cadherin (Cell Signaling Technology, Danvers, MA, USA), vimentin (Cell Signaling Technology), and β-actin (Abcam, Cambridge, UK). NIH3T3 whole cell lysate (Novus Biologicals, Centennial, CO, USA) was used as a positive control for mesenchymal markers. The ChemiDoc MP imaging system (Bio-Rad, Hercules, CA, USA) was used to obtain images, and fluorescence intensities were quantified using the ImageLab software (Bio-Rad).