Journal: Scientific Reports

Article Title: Identification of hnRNP-A1 as a pharmacodynamic biomarker of type I PRMT inhibition in blood and tumor tissues

doi: 10.1038/s41598-020-78800-6

Figure Lengend Snippet: Pharmacology of arginine methylation with type I PRMT inhibition. Schematic of arginine methylation mediated by PRMTs and mechanism of type I PRMT inhibition. The left side of the figure shows normal arginine methylation and the right side illustrates the decrease in ADMA with a concomitant increase in MMA and SDMA upon type I PRMT inhibition.

Article Snippet: 12 mg total protein for each sample was desalted over SEP PAK C18 columns and split into 3–4 mg aliquots for enrichment with MMA motif [mme-RG] immunoaffinity beads (CST, #12235), a mixture immunoaffinity beads conjugated to ADMA motif [adme-R] and custom ADMA antibody (clone D10F7A10) (CST, #13474; modified method), and SDMA motif [sdme-RG] immunoaffinity beads (CST, #13563).

Techniques: Methylation, Inhibition

Journal: Scientific Reports

Article Title: Identification of hnRNP-A1 as a pharmacodynamic biomarker of type I PRMT inhibition in blood and tumor tissues

doi: 10.1038/s41598-020-78800-6

Figure Lengend Snippet: Effects of type I PRMT inhibition in human PBMCs. Western Blot analysis of monomethyl-arginine (MMA), symmetric dimethyl arginine (SDMA), asymmetric dimethyl arginine (ADMA), PRMT1 and PRMT5 (NC, non-cultured, NS, non-stimulated, TCR act , T Cell Receptor activated) ( A ) and RT-PCR for several PRMTs ( B ) in non-stimulated and TCR-activated human PBMCs from healthy donors treated with either DMSO or 2 µM GSK3368712 for 72 h (two-tailed Student’s t test: * p < 0.05, ** p < 0.01, *** p < 0.001; n.s., not significant). ( C–E ) MethylScan analysis of arginine methylation in human PBMCs treated with GSK3368712. ( C ) Venn diagram representation of proteins modulated by the type I PRMT inhibitor at MMA, ADMA and SDMA sites across four healthy donors. ( D ) Common set of proteins where a decrease in ADMA and concomitant increase in MMA and SDMA was observed in all 4 donors. ( E ) Pathway analysis of all proteins that underwent any change (> 2.5 fold) in arginine methylation upon treatment with GSK3368712, compared to DMSO. FDR, False Discovery Rate.

Article Snippet: 12 mg total protein for each sample was desalted over SEP PAK C18 columns and split into 3–4 mg aliquots for enrichment with MMA motif [mme-RG] immunoaffinity beads (CST, #12235), a mixture immunoaffinity beads conjugated to ADMA motif [adme-R] and custom ADMA antibody (clone D10F7A10) (CST, #13474; modified method), and SDMA motif [sdme-RG] immunoaffinity beads (CST, #13563).

Techniques: Inhibition, Western Blot, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test, Methylation

Journal: Scientific Reports

Article Title: Identification of hnRNP-A1 as a pharmacodynamic biomarker of type I PRMT inhibition in blood and tumor tissues

doi: 10.1038/s41598-020-78800-6

Figure Lengend Snippet: Mass Spectrometry analysis of hnRNP-A1 immunoprecipitated from Toledo cells treated with GSK3368712. (A) Amino acid sequence 180–240 of human hnRNP-A1 (* methylated arginine residues identified by MS). ( B, C and D ) Chromatographic representation of the methylation forms found at arginine residues 194, 206 and R225 and their relative changes induced by treatment with the type I PRMT inhibitor GSK3368712 (2 µM) for 48 h. 0MA, unmethylated arginine. MMA, monomethylated arginine. DMA, dimethylated arginine. ( E, F and G ) Relative levels of asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) with respect to total dimethylarginine 194, 206 and 225 based on generation of diagnostic neutral loss fragment ions.

Article Snippet: 12 mg total protein for each sample was desalted over SEP PAK C18 columns and split into 3–4 mg aliquots for enrichment with MMA motif [mme-RG] immunoaffinity beads (CST, #12235), a mixture immunoaffinity beads conjugated to ADMA motif [adme-R] and custom ADMA antibody (clone D10F7A10) (CST, #13474; modified method), and SDMA motif [sdme-RG] immunoaffinity beads (CST, #13563).

Techniques: Mass Spectrometry, Immunoprecipitation, Sequencing, Methylation, Diagnostic Assay