Fig. 3

Characterization of FGF12 complexes with NOLC1 and TCOF1. A U2OS-FGF12-mGFP-myc and U2OS (control) cells were lysed and the co-purification of NOLC1 and TCOF1 with FGF12-mGFP-myc was determined with SDS-PAGE and western blotting. B In situ proximity ligation assay (PLA) using rabbit anti-FGF12, mouse anti-NOLC1 or anti-TCOF1 antibodies in U2OS-FGF12-mGFP-myc cells. Cell nuclei were labeled with NucBlue Live and cells were analyzed by fluorescence microscopy. The dashed line indicates the cell area. The scale bar represents 20 μm. Data shown in the graphs are mean PLA signal intensities in nuclei ± SD from three independent experiments (100 cells in total). Student’s t-test was applied for statistical analysis; ***p < 0.001. C Pull-down experiment with recombinant FGF12-His bound to Ni Sepharose and U2OS cell lysate incubated with calf intestinal alkaline phosphatase (CIP), analyzed by SDS-PAGE and western blotting using anti-FGF12, anti-NOLC1 or anti-TCOF1 antibodies. An antibody against γ-tubulin was used to ensure equal loading of input samples. D Pull-down experiment with recombinant FGF12-His bound to Ni Sepharose and nuclease-treated U2OS cell lysate, analyzed by SDS-PAGE and western blotting using anti-FGF12, anti-NOLC1 or anti-TCOF1 antibodies. An antibody against γ-tubulin was used to ensure equal loading of input samples. E Structural (based on PDB 1Q1U structure) and bar representation of FGF12 with the deleted region in FGF12Δ-His marked in red (upper panel). F Biological activity of FGF12Δ-His variant. Serum-starved NIH 3T3 cells were incubated with recombinant FGF12Δ-His (~ 1 µg/ml) or FGF1 (control, ~ 0.1 µg/ml) for 45 min, and activation of cell signaling was assessed by western blotting with antibodies specific for activated FGFR and ERK1/2 (pFGFR; pERK1/2). An antibody against γ-tubulin was used to ensure equal loading. G Interaction of FGF12Δ-His mutant with NOLC1 and TCOF1. Recombinant FGF12-His and FGF12Δ-His were bound to Ni Sepharose and incubated with U2OS cells lysate. Elution fractions were subjected to western blot analysis with anti-NOLC1, anti-TCOF1 and anti-His-tag antibodies. H Lysates of U2OS cells expressing FGF11-mGFP-myc, FGF12-mGFP-myc, FGF13-mGFP-myc, FGF14-mGFP-myc were incubated with anti-myc agarose. Co-purification of NOLC1 and TCOF1 with FHFs was determined with western blotting using anti-GFP, anti-NOLC1 and anti-TCOF1 antibodies. An antibody against γ-tubulin was used to ensure equal loading of input samples.