Journal: Nature Communications

Article Title: PRMT3-mediated arginine methylation of IGF2BP1 promotes oxaliplatin resistance in liver cancer

doi: 10.1038/s41467-023-37542-5

Figure Lengend Snippet: a Venn Diagram showing the 84 common proteins pulled down by PRMT3 in PLC-8024 and HepG2 cells which were analyzed by liquid chromatography/tandem mass spectrometry (LC–MS/MS). b The number of peptide fragments (95% CI) of proteins pulled down by PRMT3 in PLC-8024 cells and HepG2 cells identified from the IP-MS analysis. c WB analysis showed that endogenous PRMT3 and IGF2BP1 interact with each other in PLC-8024 cells using reciprocal co-immunoprecipitation. d WB analysis showed that endogenous IGF2BP1 pulled down endogenous PRMT3 in Huh7 cells, and endogenous PRMT3 pulled down endogenous IGF2BP1 in Huh7-R cells. e Immunofluorescence staining showed the co-localization of PRMT3 (green) and IGF2BP1 (red) in HCC cells and HCC patient samples. Scale bar, 50 μm. f WB analysis of immunoprecipitated IGF2BP1 to determine the effect of PRMT3 OE and PRMT3 inhibition by SGC707 on arginine methylation of IGF2BP1 in PRMT3-KO PLC-8024 cells using asymmetric dimethylarginine antibody (ADMA). g WB analysis of immunoprecipitated IGF2BP1 to determine the effect of PRMT3 KD on arginine methylation of IGF2BP1 in PLC-8024/Huh7-R cells. h The sequences surrounding R452 of IGF2BP1 are evolutionarily conserved across multiple species. i A scheme showing the sequences of Flag-tagged IGF2BP1-WT and IGF2BP1-R452K mutant. j WB analysis of immunoprecipitated Flag-tagged IGF2BP1-WT and IGF2BP1-R452K mutant showed that R452K mutation dramatically reduced ADMA signal in HEK293 cells overexpressing Flag-tagged IGF2BP1-WT and IGF2BP1-R452K mutant. For c – g , and j n = 3 independent experiments. Source data are provided as a Source Data file.

Article Snippet: The input and output samples were resolved by SDS-PAGE and detected by immunoblot analysis with the indicated primary antibodies: anti-GAPDH antibody (Proteintech, 60004-1-Ig, 1:2000 dilution), anti-PRMT3 antibody (Abcam, Ab191562, 1:2000 dilution), anti-IGF2BP1 antibody (Proteintech, 22803-1-Ap, 1:1000 dilution) and anti-ADMA antibody (Cell Signaling Technology, 13522S, 1:1000 dilution).

Techniques: Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Protein-Protein interactions, Immunoprecipitation, Immunofluorescence, Staining, Inhibition, Methylation, Mutagenesis