Phospho and total erk1 2

Paraffin-embedded human eye sections were deparaffinized, rehydrated, and fixed in acetone. Primary antibodies: IL17A (polyclonal rabbit anti-human IgG, 1∶100 dilution, Catalog #13082-1-AP, Proteintech, Chicago, IL) IL17RC (polyclonal rabbit anti-human IgG, 1∶100 dilution, Catalog #sc-99937, Santa Cruz Biotechnology, Santa Cruz, CA). Secondary antibody: biotin conjugated, goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA). Chromagen: 3,3′-diaminobenzidine (Biocare Medical, Concord, CA). Slides were counterstained with 1% methyl green. For westerns, primary antibodies include phospho- and total-Erk1/2, phospho- and total-Akt, phospho- and total-p38, and GAPDH (Cell Signaling Technology).

Medial lobe liver sections were snap frozen at the time of euthanasia. Liver tissue was homogenized in RIPA buffer containing a protease and phosphatase cocktail (Thermofisher, Waltham, MA). Samples were then prepared with laemmli sample buffer and β-mercaptoethanol at a 4 µg/µl concentration and 32 µg of total protein run down each lane of a 4–20% gradient gel (Biorad, Hercules, California) at 70 V. Samples were then transferred to 0.45 µm nitrocellulose membranes, dried, reconstituted with _ddiH₂O, blocked with LiCor (Lincoln, Nebraska) TBS blocking buffer, and incubated in primary antibody overnight at 4 °C. Primary antibodies include β-actin (Santa Cruz Biotechnology, Dallas, Texas), phospho myosin light chain (Thr18/Ser19) (Cell Signaling Technologies, Danvers, MA), phospho and total p65 (Cell Signaling Technologies, Danvers, MA), phospho and total ERK 1/2 (Cell Signaling Technologies, Danvers, MA), and catalase (Abcam, Cambridge MA). Membranes were then washed with TBS containing 1% tween (TBST), incubated in Licor near-infrared secondary antibodies for 1 h at room temperature, washed again with TBST, and finally imaged with the LiCor Odyssey Clx. Densitometry analysis was conducted in Image-J (National Institutes of Health, Bethesda, Maryland) with phosphor-signal normalized to total-signal and fold change calculated from control.

Total protein from Y- and A-TSPC (clustered EphA4 and EphB2, and Fc-control) was isolated with RIPA-buffer (0.1% SDS, 1% Na-DOC, 1% Triton X-100, 50mM Tris-HCl pH8.2, 150mM NaCl, 10mM EDTA, 20mM NaF, 1mM Na₃VO₄) supplemented with complete protease inhibitors (Roche). Proteins (20 μg) were separated on SDS gels, transferred onto PVDF membrane and blocked with 5% skim milk (Merck) for 1 h at room temperature. Primary antibodies against human EphA4 (Abnova), EphB2 (Biomol), EphB4 (Thermo scientific), EFNB1 and EFNB2 (both Sigma–Aldrich); phospho-FAK (Thermo scientific), total FAK, total and phospho-ERK1/2; total and phospho-Akt; total and phospho-p38; total and phospho-Jnk (all Cell Signaling, USA), and GAPDH (Merck) were applied overnight at 4°C. Then, membranes were incubated with corresponding secondary HRP-conjugated antibodies (Cell Signaling) for 1 h at room temperature and consequently with ECL solution (GE Healthcare, USA). Photomicrographs were taken on ImageQuant LAS 4000 mini (GE Healthcare) as band intensities were quantified with ImageProPlus4 software program (Media Cybernetics, USA). Western blot experiments were preformed two independent times with all three Y- and A-TSPC donors (n = 6).