Fig. 4: PRMT1 induces asymmetric dimethylation(ADMA) of NUSAP1 at R418 and R422, but not PRMT5.

A Mass spectrometry (MS) analysis identifying dimethylated arginine residues (R418me2 and R422me2) on NUSAP1. B Sequence alignment of NUSAP1 across multiple species highlighting the conserved arginine residues (R418 and R422) subject to methylation. C Co-IP assay performed in HEK293FT cells expressing wild-type (WT) or mutant (R418K and R422K) MYC-tagged NUSAP1, followed by Western blotting to detect asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA). Effects of PRMT inhibitors on NUSAP1 methylation. HEK293FT cells expressing MYC-NUSAP1 were treated with increasing concentrations of PRMT1 inhibitors AMI-1 (D) and DCLX069 (E), or PRMT5 inhibitor GSK3235025 (F). Co-IP was performed to assess NUSAP1 methylation. G Co-IP assay showing reduced NUSAP1 methylation in BGC-823-5-FU-R and SGC-7901-5-FU-R cells transfected with shRNAs targeting PRMT1 (shPRMT1-1# and shPRMT1-2#) compared to control cells. H Co-IP assay in BGC-823-5-FU-R and SGC-7901-5-FU-R cells co-expressing wild-type (WT) or methylation-deficient mutant (G80R) Flag-PRMT1 and MYC-NUSAP1, with or without PRMT1 knockdown. Western blotting was used to detect ADMA levels on NUSAP1.