Fig 2: Identification of FGF12 partner proteins. A Recombinant FGF12-His was bound to Ni Sepharose and incubated with U2OS cell lysate. Proteins were eluted with Laemmli sample buffer, separated with SDS-PAGE and stained with CBB. B Top 10 results of identifying proteins by MS that specifically bound to FGF12-His in pull-down experiments. ID is the Uniprot protein identifier, the abbreviated name is also the gene name. The score and peptide match for each of the two repeats of the experiment are given. C FGF12 interaction network identified by affinity purification followed by mass spectrometry. Proteins were classified using STRING database (http://www.string-db.org/). Blue spheres represent nuclear proteins and red spheres represent cytosolic proteins. The lines connecting the spheres represent the experimentally confirmed interactions. D Cellular localization of FGF12. U2OS cells stably expressing FGF12-mGFP-myc (U2OS-FGF12-mGFP-myc) were fixed, then cell nuclei were stained with NucBlue Live, and analyzed with fluorescence microscopy. The dashed line indicates the cell area and the arrows indicate the nucleoli. The scale bar represents 20 μm. The graph shows the mean fluorescence intensity in the nucleus and cytoplasm. Data presented are means ± SD of 20 cells. A paired student’s t-test was used for statistical analysis; ***p < 0.001. E Co-localization of FGF12-mGFP-myc with the nucleolar marker, dyskerin. U2OS-FGF12-mGFP-myc cells were fixed, permeabilized and stained with a primary antibody against dyskerin and Alexa Fluor 594-conjugated anti-mouse secondary antibody. Cell nuclei were labeled with NucBlue Live and cells were analyzed by fluorescence microscopy. The scale bar represents 20 μm. F U2OS-FGF12-mGFP-myc cells were lysed, and nucleoli were isolated by sonication and centrifugation in buffer with increasing sucrose concentration. The presence of FGF12 and NOLC1 in nucleoli was analyzed by immunoblotting. An antibody against γ-tubulin was used to ensure equal loading of the samples.

Fig 3: Interaction of FGF12 with nucleolar proteins. A Verification of the results of MS experiments. Recombinant FGF12-His was bound to Ni Sepharose and incubated with U2OS cells lysate. FGF12-bound proteins were eluted and analyzed by wester blotting using specific antibodies. B Co-localization of FGF12-mGFP-myc with identified partner protein. To analyze the co-localization of FGF12-mGFP-myc with NOLC1, TCOF1, BCLAF1, THRAP3, U2OS-FGF12-mGFP-myc cells were fixed, permeabilized and stained with appropriate primary antibodies and Alexa Fluor 594-conjugated anti-mouse secondary antibody. Cell nuclei were labeled with NucBlue Live and cells were analyzed by fluorescence microscopy. The scale bar represents 20 µm.

Fig 4: 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 ?-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 ?-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 FGF12?-His marked in red (upper panel). F Biological activity of FGF12?-His variant. Serum-starved NIH 3T3 cells were incubated with recombinant FGF12?-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 ?-tubulin was used to ensure equal loading. G Interaction of FGF12?-His mutant with NOLC1 and TCOF1. Recombinant FGF12-His and FGF12?-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 ?-tubulin was used to ensure equal loading of input samples.