Ma5 35295

The podocytes were lysed in RIPA cell lysis buffer (250 μL). After centrifugation at 12,000 r/min for 20 min, the protein concentration was detected using a bicinchoninic acid kit (Thermo Fisher Scientific). Then, an equal volume (50 μg per well) of protein was separated by 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Bio-Rad). The membranes were blocked with 5% skimmed milk at 20°C for 1 h, and then co-cultured with the primary antibodies against Podocin (1:1000, ab181143, Abcam), FOXA1 (1:1000, ab170933, Abcam), SATB1 (1:1000, ab109122, Abcam), β-catenin (1:5000, ab32572, Abcam), active (non-phospho), β-catenin (Ser33/37/Thr41) (1:1000, #8814, CST, Cell Signaling Technology (CST), Beverly, MA, USA), TCF4 (1:2000, MA5-35295, Thermo Fisher Scientific) and GAPDH (1:1000, #5174, CST) at 4°C overnight, and then with the secondary antibody goat anti-rabbit IgG H&L (HRP) (1:2000, ab205718, Abcam) at 20°C for 1 h. After that, the protein bands were visualized using an enhanced chemiluminescence kit (Thermo Fisher Scientific). Relative protein level was calculated using the Image J software according to the grey value ratio of target bands to the internal references.

The 4-μm sections were rehydrated, blocked with 3% H₂O₂ and placed in a microwave for antigen retrieval. Then, the sections were blocked in 5% bovine serum albumin for 1 h and hybridized with the primary antibodies at 4°C overnight, followed by the incubation with secondary antibody at 37°C for 45 min. Phosphate-buffered saline (PBS) was used instead of primary antibody as negative control. 3,3ʹ-diaminobenzidine (DAB) was used for color development, and the nuclei were counter-stained with hematoxylin. The staining was observed under the microscope. The positive cells were stained in dark brown. The optical density (OD) in each section was evaluated using the Image J. The primary sections used were Podocin (1:500, ab181143, Abcam Inc., Cambridge, MA, USA), FOXA1 (1:1000, ab170933, Abcam), SATB1 (1:100, ab109122, Abcam), β-catenin (1:5000, ab32572, Abcam), Collagen I (1:500, ab270993, Abcam), Fibronectin (1:100, ab2413, Abcam) and transcription factor 4 (TCF4; 1:50, MA5-35295, Thermo Fisher Scientific). The secondary antibody used was IgG H&L (HRP) (1:2000, ab205718, Abcam).