Wnt3a

Protein lysates of cells were prepared using NP40 lysis buffer. Then, 30 μg of proteins was separated by 8% SDS-PAGE and then transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with blocking buffer and then incubated with the primary antibodies for pAkt (#4060), Akt (#4691), pErk1/2 (#9101), Erk1/2 (#9102) (Cell Signaling Technology Danvers, MA, USA), Wnt3a (GTX128101), Wnt7a (GTX128106), Lef1 (GTX129186) (GeneTex, Taiwan) and β-actin (Sigma-Aldrich, St Louis, MO, USA), followed by incubating with secondary antibodies peroxidase-AffiniPure goat anti-mouse (115-035-003) or anti-rabbit (111-035-144) antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA).

Regenerative and inflammatory markers were analyzed using optic nerve tissue. Lysates were produced from optic nerve tissue using a PRO-PREP solution (iNtRON Biotechnology, Gyeonggido, Korea). The same amounts of total proteins were separated by SDS-electrophoresis and transferred to the membrane. The membranes were incubated with anti-Thy-1 (SC-53116), β-actin (SC-47778) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Vegf (GTX102643), Tnf-α (GTX10520), β-catenin (GTX101435), Wnt3a (GTX128101), (GeneTex, Irvine, CA, USA), or GFAP (#3670), Neurofilaments (#2837), tCaspase3 (#9662), Bcl2 (#2764), Nf-κb (#8242) (Cell Signaling Technology, Danvers, MA, USA) or Hif-1α (PA1-16601), Bdnf (PA5-85730), Iba1 (PA5-27436) (Thermo Fisher Scientific) or Nlrp3 (NBP2-12446) (Novus Biologicals, Centennial, CO, USA) or NeuN (MABB377) (Millipore) antibodies. All antibodies except Thy-1 (1:200 dilution) were used in a 1:1000 dilution ratio. After washing steps, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit or mouse secondary antibodies at a 1:10,000 dilution (GeneTex) for o/n at 4 °C. Immuno-active bands were visualized as enhanced chemiluminescence solutions (Bio-Rad Laboratories, Hercules, CA, USA) and detected using ImageQuant™ LAS 4000 (GE Healthcare, Chicago, IL, USA).