Fig. 3: NUSAP1 Binds to PRMT1 and PRMT5.
From: PRMT1-catalyzed NUSAP1 methylation enhances Notch2 signaling and 5-FU resistance in gastric cancer
A Schematic representation of the experimental workflow used to identify NUSAP1-interacting proteins. HEK293FT cells were transfected with a MYC-NUSAP1 overexpression plasmid, and NUSAP1-interacting proteins were pulled down using an anti-MYC antibody followed by mass spectrometry (MS) analysis. B Venn diagram summarizing the NUSAP1-interacting proteins identified by MS. PRMT1 and PRMT5 were highlighted as potential interaction partners of NUSAP1. C Co-immunoprecipitation (Co-IP) assay performed in HEK293FT cells to confirm the interaction between NUSAP1 and PRMT1/PRMT5. MYC-tagged NUSAP1 was immunoprecipitated, and the presence of PRMT1 and PRMT5 was detected by Western blotting. D Co-IP assay performed in BGC-823-5-FU-R and SGC-7901-5-FU-R cells to validate the interaction between endogenous NUSAP1 and PRMT1/PRMT5. E, G Co-IP assay using anti-PRMT1 or anti-PRMT5 antibodies in BGC-823-5-FU-R and SGC-7901-5-FU-R cells to detect the presence of NUSAP1. Proximity ligation assay (PLA) showing the colocalization of NUSAP1 with PRMT1 (F) and PRMT5 (H) in BGC-823-5-FU-R and SGC-7901-5-FU-R cells. Tubulin was stained as a cytoskeletal marker. Scale bar = 2 μm. I Purification of GST-tagged NUSAP1, His-tagged PRMT1, and His-tagged PRMT5 proteins, as shown by SDS-PAGE. J GST pull-down assay was performed using GST-NUSAP1,His-PRMT1 and His-PRMT5 proteins. Pulled-down proteins were analyzed by western blotting using anti-GST and anti-His antibodies.
