Norrin

TriZol was purchased from Invitrogen (NY, US). Reverse Transcription and RT-PCR System were purchased from Roche (Mannheim, Germany) and Takara (Shiga, Japan), respectively. All other reagents were obtained from Sigma (St. Louis, MO). For western blot analyses and immunocytochemistry, a rabbit polyclonal antibody against the humanWnt2b receptor (ab50575), a polyclonal antibody against human Fzd5 (ab14475) and a monoclonal antibody against β-catenin (E247) (ab32572) were obtained from Abcam (MA, US), and an antibody to rat GAPDH was obtained from Epitomics (2251-1, CA, US). The HRP-conjugated secondary antibody (sc-2004) for western blotting was from Santa Cruz Biotechnology (CA, USA). The proteins DKK-1 (a Wnt-pathway inhibitor) and Norrin (a Wnt-pathway activator) were purchased form R&D Systems (Minneapolis, MN, USA).

bEnd.3 cells, obtained from ATCC (CRL‐2299), were grown in DMEM with 4.5 g/l glucose, 3.7 g/l sodium bicarbonate, 4 mM glutamine, 10% FBS, 1% penicillin/streptomycin and cultured in 10 cm dishes until confluency. bEnd.3 cells were passaged using trypsin 0.25%‐EDTA solution at a split ratio of 1:4–1:10 and cells were used between p2 and 19 for the experiments. For cell treatment, 200,000 cells were seeded per well of a 6‐well plate. After 16 h, cells were around 60–70% confluency and were treated with Norrin (R&D, Cat# 3014‐NR), F4L5.13, and isotype control for 24 h. HEK293T cells were authenticated with STR profiling at The Centre for Applied Genomics (Toronto, Ontario, Canada). HEK293T cells were cultured in high‐glucose DMEM with 10% FBS at 37°C in the presence of 5% CO₂. For maintenance, cells were split 1:6 at near confluence using 0.05% Trypsin‐EDTA. Cells were routinely tested for Mycoplasma using the MycoAlert Plus detection kit (Lonza, LT07‐701).