Journal: The Journal of Biological Chemistry
Article Title: Fortilin binds and stabilizes MEF2C, activates it through phosphorylation, and drives transcription of the cell structural and survival protein CTNNA3
doi: 10.1016/j.jbc.2026.111417
Figure Lengend Snippet: Fortilin physically binds MEF2C. A, quantitative RT–PCR (qRT–PCR) analysis of THP1 WT-fortilin and THP1 KO-fortilin cells ( n = 3, p < 0.0001, two-sample t test). B – E, MST to characterize the interaction of fortilin with MEF2 family proteins, MEF2A, MEF2B, MEF2C, and MEF2D. K d s were calculated using NanoTemper Analysis software based on the relative F norm values. F – I, in vivo forward co-IP assays. Fortilin was immunoprecipitated from the TCLs of 293T cells using an α-fortilin Ab. Successful IP of fortilin and co-IP of MEF2C and other MEF2 isoforms were assessed by Western blotting using α-fortilin Ab and Abs against MEF2A, MEF2B, MEF2C, and MEF2D, respectively. Total protein was visualized in the SDS-gel containing TCE. J, in vivo reverse co-IP assay. MEF2C was immunoprecipitated from the 293T TCL using an α-MEF2C Ab. Successful IP of MEF2C and co-IP of fortilin were assessed by Western blotting using α-MEF2C and α-fortilin Abs, respectively. Total protein was visualized in an SDS-gel containing TCE. K and L, PLA to evaluate the fortilin–MEF2C interaction. Phorbol 12-myristate 13-acetate-treated THP1 WT-fortilin and THP1 KO-fortilin cells cultured on chamber slides were subjected to a standard PLA assay using rabbit α-fortilin and mouse α-MEF2C Abs. Nuclei were counterstained by DAPI ( blue ). Red puncta indicated that fortilin and MEF2C were located within 30 nm of each other. The scale bar represents 50 μm. K, the PLA interaction index was calculated as the number of PLA puncta per nucleus within a field of view and expressed in AU. Three fields were quantified for each sample, and three independent experiments were performed. Statistical significance was evaluated using a Welch’s two-sample t test ( L ). Ab, antibody; AU, arbitrary unit; DAPI, 4′,6-diamidino-2-phenylindole; α-FLAG, anti-FLAG (DYKDDDDK) antibody (Ab); F norlm , normalized fluorescence value; α-fortilin, rabbit anti-fortilin monoclonal Ab; Fortilin KO, THP1 cells in which the fortilin ( TPT1 ) genes have been deleted by the CRISPR–Cas9 technology (THP1 KO-fortilin ); Fortilin WT, THP1 cells expressing WT fortilin (THP1 WT-fortilin ); α-His 6 , anti-hexahistidine Ab; IB, immunoblot; IgG, normal rabbit immunoglobulin G; INPUT, 10% of TCLs used for IP; IP, immunoprecipitation; α-MEF2C, anti-MEF2C Ab; MST, microscale thermophoresis; PLA, proximity ligation assay; TCE, 2,2,2-trichloroethanol; TCL, total cell lysate.
Article Snippet: Briefly, we harvested THP1 KO-fortilin and THP1 WT-fortilin cells, isolated total RNA using the GeneJET RNA Purification Kit (Thermo Fisher Scientific, catalog no.: K0731), and subjected 50 ng of total RNA to real-time RT–qPCR using the QuantiNova Probe RT–PCR kit (QIAGEN, catalog no.: 208354) with the following primer and probe sets (Integrated DNA Technologies).
Techniques: Quantitative RT-PCR, Software, In Vivo, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, SDS-Gel, Cell Culture, Fluorescence, CRISPR, Expressing, Microscale Thermophoresis, Proximity Ligation Assay
