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. 2022 Aug 8;11(1):45. doi: 10.1038/s41389-022-00421-7

Fig. 1. HBP1 is methylated by PRMT1 at arginine 378.

Fig. 1

A Endogenous HBP1 undergoes MMA modification. Endogenous HBP1 immunoprecipitates from HeLa cells were immunoblotted with anti-HBP1 and anti-MMA antibodies. B HBP1 is methylated by PRMT1 in vivo. HA-HBP1 with FLAG-PRMT1, 3, 5, 6 individually were co-transfected into HEK293T cells. Then total cell lysates were immunoprecipitated with anti-HA antibody and detected by western blotting. C Knock down PRMT1 decreased HBP1 methylation. Endogenous HBP1 immunoprecipitates in HeLa cells with PRMT1 knock down and then blotted with anti-MMA antibody and anti-DMA antibody. D HBP1 is methylated by PRMT1 in vitro. Purified His-HBP1 was incubated with or without GST-PRMT1 in 60 μL of HMT buffer at 37 °C for 2 h and followed by western blotting with His, GST, MMA or DMA antibodies. E, F PRMT1 interacts with HBP1 in vivo. E HEK293T cells were co-transfected with HA-HBP1 and FLAG-PRMT1. The Co-IP assay was carried out by using anti-HA/FLAG antibody and followed by western blotting with anti-FLAG/HA antibody. F Endogenous HBP1 or PRMT1 immunoprecipitates from HeLa cells were immunoblotted with anti-HBP1 or anti-PRMT1 antibodies. G PRMT1 co-localizes with HBP1 in nucleus. HEK293T cells were transfected with HA-HBP1 and stained with PRMT1 antibody (red) and anti-HA antibody (green). DAPI (blue), nucleus. Scale bar, 5 µm. H PRMT1 interacts with HBP1 in vitro. GST pull-down assays were performed using His-HBP1 /His-PRMT1 with GST or GST-PRMT1/GST-HBP1 followed by immunoblotting with anti-His antibody. I–M PRMT1 methylates HBP1 at arginine 378. I HBP1 methylation is analyzed by mass spectrometry. J Evolutionary conservation of the HBP1 R378 residue in other species. K HEK293T cells were co-transfected with HA-HBP1/R378A with FLAG-PRMT1/G98R and subjected to immunoprecipitation followed by western blotting. L, M HBP1 methylation was verified by polyclonal antibody specific for HBP1 R378me1a.