Fig. 6: NUSAP1 binds to the PEST domain of Notch2 in the cytoplasm.
From: PRMT1-catalyzed NUSAP1 methylation enhances Notch2 signaling and 5-FU resistance in gastric cancer
A Co-IP assays performed in BGC-823-5-FU-R and SGC-7901-5-FU-R cells showing interaction between NUSAP1 and Notch2. Anti-Notch2 antibody was used for immunoprecipitation, and the presence of NUSAP1 was detected by Western blotting. B Co-IP assays using anti-MYC antibody to confirm the interaction between MYC-tagged NUSAP1 and endogenous Notch2 in BGC-823-5-FU-R and SGC-7901-5-FU-R cells. C PLA showing colocalization of MYC-NUSAP1 and Notch2 in BGC-823-5-FU-R and SGC-7901-5-FU-R cells. NUSAP1 and Notch2 were detected with PLA (red) and tubulin (green) as a cytoskeletal marker. Scale bar = 2 μm. D SDS-PAGE showing the purification of His-tagged Notch2 intracellular domain (N2ICD) protein. E GST pull-down assay was performed using GST-NUSAP1 and His-tagged N2ICD proteins. Pulled-down proteins were analyzed by Western blotting using anti-GST and anti-His antibodies. F Diagram of the Notch2 intracellular domain (N2ICD) with functional regions: RAM, AR, TAD, and PEST. Interaction of these regions with NUSAP1 was assessed and is indicated by colored circles. G Co-IP assays in BGC-823-5-FU-R and SGC-7901-5-FU-R cells showing that the PEST domain of Notch2 interacts with NUSAP1. Flag-tagged N2ICD fragments (RAM, AR, TAD, PEST) were immunoprecipitated using anti-MYC antibody, and NUSAP1 was detected by Western blotting. H PLA showing colocalization of MYC-NUSAP1 and Flag-PEST domain in BGC-823-5-FU-R and SGC-7901-5-FU-R cells. Scale bar = 2 μm.
