Fig 1: GDNF-RET signaling tunes branching in mouse and human ureteric bud tissues.A. GDNF-RET signaling interactions involved in kidney branching.B. Immunofluorescence of cleared E14 mouse kidney. Top left, ECAD and SIX2. Bottom left, RET and GFRA1. Top right, isolated RET. Bottom right, isolated GFRA1 signal.C. Air-liquid interface (ALI) culture of mouse embryonic kidney explants.D. Design of GDNF-RET signaling perturbation experiments in ALI culture.E. Immunofluorescence of E13 kidneys grown in ALI culture for 4 days in 100 nM Selpercatinib (+RETi), 100 ng ml−1 GDNF (+GDNF), or control media. Inset, close-up of tip domains. Explants are immunostained for EpCAM (epithelium), RET (tip cells), and JAG1 (early nephrons).F. Tip number on days 1-4 across all conditions, N = 4 kidneys per condition. P-values by one-way Kruskal-Wallis test with Dunn’s post hoc test.G. Differentiation of iUB organoids from hiPSCsH. Immunofluorescence of epithelial (ECAD) and tip (RET) markers in a day 12 iUB organoid.I. Design of GDNF-RET signaling perturbations for iUB organoids.J. Merged brightfield and GATA3:mCherry images of iUB organoids at days 9 and 12. Organoids were cultured in control (−GDNF) or complete branching medium (+GDNF, 50 ng ml−1), excess GDNF (++GDNF, 250 ng ml−1), or complete branching medium with 100 nM Selpercatinib (+RETi). See: Fig. S4F.K. Projected area (x104 μm2) on days 9 and 12 for all conditions, n = 47, 56, 40, 46 iUB organoids (−GDNF, +RETi, +GDNF, ++GDNF) from 2 independent biological replicates.L. Circularity (a.u.) on days 9 and 12.M. Bud number on days 9 and 12. P-values in panels K, L, M by one-way ANOVA with Dunnett’s post hoc test using +GDNF as reference group.
Fig 2: Pralsetinib potently inhibits RET activation in ER+ breast cancer cell lines.A mRNA levels of RET, GFRA1 and GDNF in CCLE and ICLE. B–G Western blotting for RET expression and downstream target phosphorylation in the indicated breast cancer cell lines. H–J Western blotting (H) with quantification (I, J) of phosphorylation of RET and downstream targets following pralsetinib dose-response treatment in MCF-7 cells. Relative abundance was calculated by normalization to intensity of β-actin measured in ImageJ. GDNF treatment: 100 ng/ml, 30 min. Recombinant GFRα1 treatment: 10 ng/ml, 30 min. Pralsetinib treatment: 500 nM unless otherwise indicated in dose-response experiments, 16 h. Vehicle, distilled water for GDNF, PBS for GFRα1, and DMSO for Pralsetinib. EV: empty vector transfected cells. RET-OE: RET-overexpressing cells. shNTC; cells transfected with non-targeting control shRNA. shRET; cells transfected with shRNA targeting RET. Protein weight indications (kDa) are based on PageRuler Plus Prestained protein ladder.
Supplier Page from R&D Systems, a Bio-Techne Brand for GFR alpha-1/GDNF R alpha-1 Fc Chimera Protein
Available conjugates: Sizes Available: 100 ug