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

Fig. 2. HBP1 methylation by PRMT1 decreases HBP1 stability by promoting its ubiquitination.

Fig. 2

A, B PRMT1 reduces HBP1 protein levels. Western blot analysis of HBP1 in HeLa and MGC803 cells transfected with empty vector or FLAG-PRMT1 (A, left and middle panels)/shPRMT1 (B, left and middle panels). mRNA expression of endogenous HBP1 measured by real-time PCR in HeLa and MGC803 cells transfected with empty vector or FLAG-PRMT1 (A, right panels)/shPRMT1 (B, right panels) (n = 3, Student’s t-test). C, D PRMT1 decreases HBP1 protein stability. Western blot of endogenous HBP1 expression in empty vector- or FLAG-PRMT1(C)/shPRMT1(D)-transfected HeLa cells when treated with CHX for 0, 30, 60, or 90 min (n = 3, Student’s t-test). E PRMT1-mediated HBP1 methylation decreases HBP1 protein stability. The stable expression of wild-type HBP1 and mutant R378A were detected by western blotting in transfected HeLa cells when treated with CHX for 0, 30, 60, or 90 min (n = 3, Student’s t-test). The half-life of HBP1 protein t1/2 measurements were performed as described previously [11]. F HBP1 protein level is elevated in the presence of MG132. HeLa cells were treated with MG132 for 6 h, the protein level of HBP1 was measured by western blotting. G PRMT1 does not decrease HBP1 protein level in the presence of MG132. HeLa cells were transfected with empty vector or FLAG-PRMT1 and incubated with or without MG132 for another 6 h. The protein level of HBP1 was detected by western blotting. H–L PRMT1-mediated methylation of HBP1 promotes its ubiquitination. H HEK293T cells were co-transfected FLAG-HBP1 with or without His-ubiquitin and then immunoprecipitated with anti-FLAG antibody. The HBP1 ubiquitination was measured by western blotting with anti-multi ubiquitin antibody. I HEK293T cells were co-transfected FLAG-HBP1, His-ubiquitin with or without HA-PRMT1 and then immunoprecipitated with anti-FLAG antibody. The HBP1 ubiquitination was measured by western blotting with anti-multi ubiquitin antibody. J HEK293T cells were co-transfected His-ubiquitin with HA-HBP1 or HA-R378A and then immunoprecipitated with anti-HA antibody. The HBP1 ubiquitination was measured by western blotting. K HEK293T cells were co-transfected HA-HBP1, His-ubiquitin with or without FLAG-MDM2 and then immunoprecipitated with anti-HA antibody. The HBP1 ubiquitination was measured by western blotting with anti-multi ubiquitin antibody. L HEK293T cells were co-transfected His-ubiquitin, MDM2, HA-HBP1 or HA-R378A with or without FLAG-PRMT1 and then immunoprecipitated with anti-HA antibody. The HBP1 ubiquitination was measured by western blotting. Date were the mean ± SD. *p < 0.05, **p < 0.01.