*4.4. Neurological Deficit Assessment*

A neurological examination of experimental animals was performed in accordance with the modified Bederson method [51] 24 h after MCAO or sham surgery by a blinded researcher. The scoring criteria are as follows: score 0: no deficit; score 1: lost forelimb flexion; score 2: as for 1, plus decreased resistance to lateral push; score 3: unidirectional circling; score 4: longitudinal spinning or seizure activity; score 5: no movement.

#### *4.5. Infarction Volume Assessment*

The infarction volume was assessed by TTC staining of consecutive 2.0 mm coronal sections from bregma +4 mm to −6.0 mm. The sections were immersed into 2% TTC (T8877, Sigma Aldrich, St. Louis, MO, USA) solution for 30 min at 37.0 ◦C. The sections were transferred into 4% paraformaldehyde solution for 24 h. The sections were then photographed and analyzed using the ImageJ software (ImageJ, National Institute of Health, Bethesda, MD, USA). The infarction volume was calculated using the following formula [50]: (contralateral volume – non-infarct ipsilateral volume)/contralateral volume × 100%. The contralateral volume refers to the opposite brain hemisphere of the infarcted side.

#### *4.6. Western Blotting*

Frozen samples were homogenized for protein extraction in lysis buffer (ProPrep; Intron Biotechnology, Pyeongtaek, South Korea) with phosphatase inhibitors (Phosstop; Roche, Mannheim, Germany). Supernatants were collected from homogenized samples that were centrifuged at 15,000 rpm for 15 min at 4 ◦C. Total protein concentrations were measured using a spectrophotometer (Nano Drop ND-1000, NanoDrop Technologies Inc., Wilmington, DE, USA) and equal amount of protein (30 mg per well) were resolved on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane (Merck Millipore, Darmstadt, Germany) for 2 h. Phospho-proteins were detected by immunoblotting prior to their corresponding total protein levels, which were detected after the membrane had been stripped. Transferred proteins on membranes were fixed using 0.05% glutaraldehyde in Tris-buffered saline containing 0.05% Tween-20 (TBST) for 15 min at room temperature. The membrane was stained with Ponceau S (P7170, Sigma-Aldrich, St. Louis, MO, USA) for visualization of the transferred proteins and cut into strips according to the size of the target protein to minimize interactions between antibodies. Membranes were blocked with 5% bovine serum albumin (BSA) dissolved in TBST for 1 h at room temperature. The membranes were incubated overnight with primary antibodies diluted in 5% BSA in TBST at 4 ◦C. Rabbit was the host of all primary antibodies used for immunoblotting. The following antibodies were used—anti-Wnt1 (ab15251, 1:1000, Abcam, Cambridge, UK), anti-Wnt3 (ab32249, Abcam), anti-PORCN (ab105543, Abcam), anti-β-catenin (#9562, 1:3000, Cell Signaling Technology, Beverly, MA, USA), anti-Akt (#4691, 1:3000, Cell Signaling Technology), anti-Phospho-Akt (#9271, 1:1000, Cell Signaling Technology), anti-GSK3-β (#9315, 1:3000, Cell Signaling Technology), anti-Phospho-GSK3-β (#9336, 1:1000, Cell Signaling Technology), anti-pβ-catenin (#9561, 1:1000, Cell Signaling Technology), anti-tankyrase 1 (MBS8531631, 1:1000, MyBioSource, San Diego, CA, USA), anti-IL-1β (AB1832P, 1:1000, Sigma Aldrich), anti-IL-6 (ARC0062, 1:500, Invitrogen, Carlsbad, CA, USA), anti-IL-8 (MBS9385550, 1:500, MyBioSource), anti-TNF-α (AAR33, 1:1000, Bio-Rad, Hercules, CA, USA) and anti-β-actin (#4967, 1:10,000, Cell Signaling Technology). After overnight incubation of primary antibodies, the membranes were incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibody (#7074, 1:10,000, Cell Signaling Technology). Visualization of immunoreactive proteins was performed with the application of chemiluminescent detection reagent (ECL™ Prime, GE Healthcare, Little Chalfont, UK) and images were taken by ImageQuant™ LAS 4000 (GE Healthcare). Protein immunoreactivity was measured using Multi-gauge software (Fuji Film Inc., Tokyo, Japan).


**Table 1.** Primer pairs for qPCR.

#### *4.7. qPCR*

RNA was extracted by using the Hybrid-R kit (305-010; GeneAll Biotechnology, Seoul, Korea). The concentration of RNA was measured using a spectrophotometer (Nano Drop ND-1000, NanoDrop

Technologies Inc.). cDNA was prepared from 1 μg of total RNA using the PrimeScript 1st strand cDNA synthesis kit (Takara Bio, Shiga, Japan). PCR amplification was executed using the SYBR-Green reagent (Takara Bio) in the ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA). PCR amplification was performed in 20 μL reaction volumes. Sequences for oligonucleotide primers were selected using the Gene Database of National Center for Biotechnology Information (NCBI) and Primer Express™ Software v3.0.1 (Thermo Fisher Scientific, Waltham, MA, USA). Primer pairs are listed in Table 1.
