Af212

Briefly, cells were harvested and lysed in RIPA lysis buffer containing protease inhibitor cocktail (11836145001, Roche) and phosphatase inhibitor cocktail (78420, Thermo Scientific). Cleared samples with 6x SDS loading buffer were denatured by boiling and resolved by SDS-PAGE. Similarly, tissue samples were first minced thoroughly and then electronically homogenized in RIPA buffer followed by 2 rounds of sonication (25% amplitude/power, 10 s) before centrifuge and SDS-PAGE separation. Antibodies used for Western blotting, Co-IP and Integrin signaling blocking include mouse anti-ACTB (1:5000, A5316, Sigma), rabbit anti-GAPDH (1:5000, 10494-1-AP, Proteintech), mouse anti-FLAG (1:2000, F1804, Sigma), rabbit anti-Ism1 (1:5000, Genescript), mouse anti-V5 (1:3000, R960-25, Invitrogen), rabbit anti-phosphorylated FAK (1:1000, #3283, CST), rabbit anti-phosphorylated AKT (1:1000, #9271, CST), rabbit anti-phosphorylated ERK (1:1000, #4370, CST), rabbit anti-phosphorylated SRC (1:1000, #2101, CST), mouse anti-N-cadherin (1:2000, #610920, BD), Goat anti-Gdnf (1:2000, AF212, R&D), HRP-conjugated mouse (1:10000, GE Healthcare, NA9310) and HRP-conjugated rabbit (1:10000, GE Healthcare, NA9340).

Cryosections of embryonic kidney rudiments were processed for proximity ligation assay as previously described^{66 (link)}. For the PLA labelling, probe anti-goat PLUS (Sigma, Duolink DUO92003), probe anti-rabbit MINUS (Sigma, Duolink DUO92004), and Duolink in situ detection reagent red (Sigma, Duolink DUO92008) were used. The following primary antibodies were used: rabbit anti-Ret antibody (1:200, Abcam, ab134100) goat anti-GDNF (1:400, R&D, AF212), rabbit anti-Ism1 antibody (1:300, Genescript) and goat anti-Integrin α8 (1:400, AF4076, R&D). Sections were mounted in the mounting medium with DAPI and subject to photograph using Zeiss LSM 800 confocal microscope.