T caspase3

Ice‐cold radioimmunoprecipitation assay buffer (containing 50 mmol/L Tris‐Hcl, 150 mmol/L NaCl, 1% Triton X‐100, 1% sodium deoxycholate and 0.1% SDS) was used to extract protein from cardiomyocytes and heart tissue. Then protein was subjected to 10% SDS‐PAGE (50 μg per sample). After transferred onto immobilon membranes (Millipore, Billerica, MA, USA), proteins were incubated with primary antibodies overnight at 4°C. The primary antibodies included the following: ZBTB20 (#ab243143, Abcam, 1:1000 diluted), Bax (#2772), Bcl‐2 (#2870), c‐caspase3 (#9664), T‐caspase3 (#9661), TNFα (#11948), Phospho (P)‐ASK1 (Thr845), total (T)‐ASK1(Thr845)(# #3765), P‐JNK1/2 (#4668p), T‐JNK1/2(#9258), P‐ERK1/2 (#4370P), T‐ERK1/2 (#4695), P‐P38 (#4511P), T‐P38 (#9212P) and GAPDH (#2118, Cell Signaling Technology, 1:1000 diluted), And then incubated with second antibodies of either goat anti‐rabbit IgG (926‐32211; LI‐COR) or goat antimouse IgG (C11026‐03; LI‐COR) for one hour. Analysis and quantification was performed by an Odyssey infrared imaging system (LI‐COR Biosciences). The GAPDH was used as reference.

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).