Fig 1.
(A) Schematic presentation of the NanoLuc-conjugated FGF2. The structure of [C95S]FGF2 was adopted from the previously published FGF2 crystal structure (PBD ID: 2BFH) [22]. Positions of the Cys residues, Y32, and Y111 in FGF2 are labeled and their side-chains are shown. (B) The previously solved crystal of structure of FGF2 in complex with receptor FGFR1 and heparin (PBD ID: 1FQ9) [24]. FGF2 molecules are shown in red and the positions of their Cys residues, Y32, and Y111 are labeled.
Fig 2.
(A) SDS-PAGE analysis of 6×His-FGF2 expression in E. coli. Lane (-), before IPTG induction; lane (+), after overnight IPTG induction; lane (s), supernatant of the cell lysate after sonication; lane (p), pellet of the cell lysate after sonication. The loading amount in each lane was equal to the E. coli cells from 50 μl of the culture broth. After electrophoresis, the gel was stained with Coomaissie Brilliant Blue R250. The band representing 6×His-FGF2 is indicated by an asterisk. (B) SDS-PAGE analysis of the purified wild-type or mutant FGF2s. After elution from a heparin-Sepharose column, 1 μg of the purified protein was loaded onto the non-reducing SDS-gel. Lane 1, 6×His-FGF2; lane 2, 6×His-[C95S]FGF2; lane 3, 6×His-[Y32A]FGF2; lane 4, 6×His-[Y111A]FGF2. After electrophoresis, the gel was stained with Coomaissie Brilliant Blue R250. (C) Receptor activation assays of the purified wild-type or mutant FGF2s. HEK293T cells were transfected with the SRE-controlled NanoLuc reporter vector. After being serum starved for 12 h, the transfected cells were treated with the wild-type or mutant FGF2s for 4 h and then lysed for bioluminescence measurement. The measured data were expressed as mean ± SE (n = 3) and fitted to sigmoidal curves using the SigmaPlot10.0 software. (D) SDS-PAGE analysis of the FGF2-Luc conjugate. Lane 1, 6×His-[C95S]FGF2 used for conjugation; lane 2, the washed fraction; lane 3, the eluted fraction. Samples were loaded onto a non-reducing SDS-gel and the gel was silver stained after electrophoresis. The band representing FGF2-Luc is indicated by an asterisk.
Fig 3.
Binding of FGF2-Luc with the overexpressed FGFR1.
Living HEK293T cells transiently overexpressing human FGFR1 (transcript variant 2) were used as the receptor source. (A) Saturation binding of FGF2-Luc with the overexpressed FGFR1. Nonspecific binding data were obtained by competition with 250 nM of 6×His-FGF2. The measured bioluminescence data were expressed as mean ± SE (n = 3). The total binding data were fitted to Y = BmaxX/(Kd + X) + NsX, specific binding data to Y = BmaxX/(Kd + X), and nonspecific binding data to a linear curve. Inner panel, Scatchard plot of the specific binding data. (B) Competition binding of wild-type or mutant FGF2s with the overexpressed FGFR1 using FGF2-Luc as a tracer. The measured bioluminescence data were expressed as mean ± SE (n = 3) and fitted with sigmoidal curves using the SigmaPlot10.0 software.
Fig 4.
Binding of FGF2-Luc with the endogenous FGF receptor.
Untransfected HEK293T cells were used as the receptor source. (A) Saturation binding of FGF2-Luc with the endogenous FGF receptor. Nonspecific binding data were obtained by competition with 250 nM of 6×His-FGF2. The measured bioluminescence data were expressed as mean ± SE (n = 3). The total binding data were fitted to Y = BmaxX/(Kd + X) + NsX, specific binding data to Y = BmaxX/(Kd + X), and nonspecific binding data to a linear curve. Inner panel, Scatchard plot of the specific binding data. (B) Competition binding of wild-type or mutant FGF2s with the endogenous FGF receptor using FGF2-Luc as a tracer. The measured bioluminescence data were expressed as mean ± SE (n = 3) and fitted with sigmoidal curves using the SigmaPlot10.0 software.