Anti lef1

Total NZCs were isolated from E14.5 and E17.5 ICR mice by enzymatic digestion as previously described7 (link). For isolation of NZCs from E17.5 and P0 conditional mutants, control and mutant kidneys were sorted based on size and GFP expression (Six2-cre-EGFP and Cited1-creER^T2-EGFP), and confirmed by genotyping. Enrichment for CITED1+ cells and purification (referred to as NPCs) was performed by negative depletion with magnetic activated cell sorting using phycoerythrin-conjugated antibodies and anti-phycoerythrin Microbeads according to the manufacturer's protocol (Miltenyi Biotec)2 (link)11 (link). Purified NPCs were cultured in monolayer in keratinocyte serum-free media (KSFM, Thermo Fisher Scientific) supplemented with rh-FGF2 (50 ng ml⁻¹, R&D Systems) and 100 U ml⁻¹ penicillin–streptomycin in plates coated with human plasma Fibronectin (100 μg ml⁻¹, EMD Millipore). The identity of purified NPCs from ICR and conditional mutant mice was verified by immunostaining using anti-CITED1 (1:200, Cell Signaling), anti-SIX2 (1:200, Proteintech) and anti-LEF1(1:100, Cell Signaling) antibodies, and RT–qPCR analysis of cap mesenchyme and cortical interstitium markers before and after growth factor/inhibitor treatments.

Total protein was extracted with RIPA lysis buffer. Nuclear protein was extracted using the Nuclear-Cytosol Extraction kit (Applygen Technologies, Beijing, China). Equal amount of denatured proteins (30 µg) were separated by 12% sodium dodecyl sulfonate/polyacrylamide gel electrophoresis and transferred on to a polyvinylidene difluoride membrane. The membrane was incubated in 5% dry skim milk at room temperature (RT) for 1 h, and subsequently with anti-KMT2D (C-Term, a smaller C-terminus fragment which separates at ∼75 kDa, 1:1000, ABE206; Millipore, U.S.A.), anti-β-catenin (1:1000, 8480S; CST, U.S.A.), anti-H3K4me1 (1:1000, GTX54199; GeneTex), anti-H3K4me3 (dilution 1:1000, GTX128954; GeneTex), anti-Lef1 (dilution 1:1000, 2230S; Cell Signaling Technology), anti-Lgr4 (1:1000, sc-390630; Santa Cruz, U.S.A. ), GAPDH (1:10000, 10494-1-AP; Proteintech, China) and anti-LaminB1 (1:5000, 66095-1-Ig; Proteintech) separately at 4°C overnight. Antibody recognition was detected with horseradish peroxidase-coupled goat anti-rabbit IgG (1:5000, ZB-5301; ZSGB-BIO, China) or goat anti-mouse IgG (1:5000, ZB-5305; ZSGB-BIO, China) for 1 h at RT. GAPDH was used as an internal control for total protein detection, and LaminB1 for nuclear protein. The presentative images of three independent experiments were presented.