Fig. 3

Identification of MDH2 and PSMA1 as the binding proteins for MDHDH. A Predicted secondary structure of MDHDH generated by the NONCODE built-in online tool. Some of the break-points of fragments used in pulldown were marked (arrowheads) with nucleotide numbers: 1–293, 294–564, 565–749, 1-49/495-577, 50-494 and 578-749. Each nucleotide is color-coded. B Schematic illustration of the RNA pulldown assay followed by mass spectrometry analysis. C Venn diagram illustrating the eluted proteins of the MDHDH full-length sense RNA probe and antisense RNA probe detected by mass spectrometry analysis (sense: 238, antisense: 275, sense-only: 37). D Metascape enrichment analysis and PPI network for the list of proteins that specifically bind to the full-length sense RNA probe (upper). Pathway enrichment results (bottom). E Molecular complex detection (MCODE) was applied to explore the significant modules in the PPI network. MCODE and GO results and description (bottom). F Western blot analysis indicating that MDH2 was regulated by MDHDH. qRT–PCR results indicate that the expression level of MDH2 was not affected by the overexpression of MDHDH. The regulation of MDH2 by MDHDH is a posttranscriptional regulation. G Western blot analysis showing the interaction between MDHDH, MDH2 and PSMA1. MDHDH antisense eluted protein was used as a negative control. H, RIP assays were performed with U87 and U251 cell extracts using anti-MDH2, anti-PSMA1 or mouse IgG. IgG served as the negative control. RNAs enriched in anti-MDH2, anti-PSMA1 and IgG pulldowns were determined relative to the input control. Agarose gel electrophoresis of RIP-PCR products (bottom). I FISH probe of MDHDH (red) costained with MDH2/PSMA1 (green) fluorescence. Fluorescence assessment of MDHDH colocalized (yellow) with MDH2/PSMA1