Figure 1.

Mammalian Fng proteins have differential effects on ligand-induced Notch1 signaling. (A) 3T3 cells were cotransfected with Notch1 (N1) and either SEAP, LFng-AP (LFng), MFng-AP (MFng), or RFng-AP (RFng), along with CBF-luciferase reporter pGL3JH26 and Renilla luciferase reporter pRLTK constructs; cocultured with either L, D1, or J1 cells; and assayed for luciferase activity. Luciferase activity is expressed as percent fold activation reflecting normalized relative luciferase units (RLUs) induced by ligand-expressing cells over RLUs obtained with L cells (ligand-activated N1+SEAP RLUs are set to 100% [D1/L = 8.65 ± 0.7, J1/L = 9.54 ± 1.5), bar graph shows mean ± SD; ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, n = 6; result from three independent experiments, each experiment done in duplicate). (B) 293T cells were either mock transfected (Mock) or transfected with SEAP, LFng-AP (LFng), LFngADD-AP (LFADD), RFng-AP (RFng), or RFngADD-AP (RFADD) plasmids. Conditioned medium and whole cell lysates from transfected cells were collected 48 h later and assayed for alkaline phosphatase (AP) activity and expressed as absorbance at OD₄₀₅ normalized for protein concentration. (C) 3T3 cells were cotransfected with Notch1 (N1) and either SEAP, LFng-AP (LFng), or LFngADD-AP (LFADD) (left) or with SEAP, RFng-AP (RFng), or RFngADD-AP (RFADD) (right) along with CBF-luciferase reporter pGL3JH26 and Renilla luciferase reporter pRLTK constructs, cocultured with either L, D1, or J1 cells and assayed for luciferase activity. Luciferase activity is expressed as fold activation over L cells reflecting normalized RLUs induced by ligand-expressing cells over RLUs obtained with L cells. Bar graph shows mean ± SD, n = 4, RLU obtained with L cells arbitrarily = 1; representative result of two independent experiments.